Abstract <p>The spleen is a vital secondary lymphoid organ present in the peritoneal cavity that plays a central role in immune surveillance and haematopoiesis. In mice, the spleen’s microanatomy is complex and differs significantly from that of humans and other domestic animals, particularly in the organisation of marginal zone and the distribution of megakaryocytes in the red pulp. Seven adult female BALB/c mice were randomly selected for the study and the standard histological procedures were employed. The immunohistochemical localisation of CD3 and CD20 were done to study the immune cell distribution in spleen of mice. The spleen of mice is intermediate type with moderate distribution of both red and white pulp as it contained moderately developed capsule, trabeculae and dual circulation. The marginal zone, peripheral to white pulp was rich in lymphocytes and prominent macrophages. The megakaryocytes were large hematopoietic cells with multilobulated nucleus, abundant pale eosinophilic cytoplasm with extensions and were commonly found in mice. This article contributes the gross and histological architecture of the murine spleen with an emphasis on the structural and functional implication of the marginal zone and the role of megakaryocytes. Immunohistochemistry stained for CD3 and CD20, confirmed clear compartmental separation of T and B cell regions, highlighting PALS as CD3-rich and follicles as CD20-rich areas. Our findings contribute to the broader understanding of localisation and distribution of different lymphoid cells involved in immune regulation which provide a roadmap for future investigations.</p> Lay Summary <p>The spleen is an important organ that helps the body fight infections and maintain healthy blood. In mice, the spleen is widely used by scientists as a model to understand how the immune system works. Studying the structure of the spleen helps researchers interpret many immunological experiments, including studies related to vaccines, infections and immune diseases. The present study aimed to examine the structure of the mouse spleen in detail, with particular attention to its cellular organization and the distribution of important immune cells. In this study, spleens from adult female BALB/c mice were examined using anatomical, histological and immunohistochemical techniques. The spleen was found to be a small, dark red organ located in the left side of the abdominal cavity. Microscopically, it consisted of two main regions called the red pulp and white pulp, which were separated by a specialized area known as the marginal zone. Each of these regions has distinct roles in immune defense and blood filtration. The red pulp contained a large number of blood-filled spaces and different blood cells. Notably, several megakaryocytes, large cells responsible for platelet formation, were observed in this region, indicating that the spleen can also participate in blood cell production outside the bone marrow. The white pulp consisted mainly of immune cells. Using specific markers, T lymphocytes were found primarily around the central arteries, while B lymphocytes were concentrated within lymphoid follicles. Overall, the study highlights the highly organized structure of the murine spleen and explains how its different compartments contribute to blood filtration, immune responses and blood cell production. These findings provide useful baseline information for future research involving mouse models in immunology and biomedical sciences.</p>