Background <p>In patients with primary hyperparathyroidism (pHPT), the importance of oxyphil cells for hormonal activity is uncertain.</p> Methods <p>This retrospective observational study analyzed the histopathological findings of postoperative specimens from patients with pHPT (single gland disease). The indication for surgery was based on increased plasma intact parathyroid hormone (iPTH) concentrations and the presence of symptoms typical of pHPT. To quantify approximate oxyphil cell content (OCC), we determined the percentage of oxyphil cells observed in each specimen. To quantify the approximate lesion volume, we determined the diameters of the fresh specimen (length, width, and height). Multiplying the lesion volume by the approximated relative OCC yielded an estimate of absolute OCC. Approximated absolute and relative OCC were then compared with preoperative plasma iPTH and calcium concentrations. Associations between these parameters were calculated using logistic or linear regression analysis.</p> Results <p>We studied 76 patients with pHPT. Increasing approximate relative OCC predicted slightly lower calcium concentration (indirect non-linear association, adjusted estimate 0.71, 95% confidence interval (CI) 0.19–1.40, <i>p</i> = 0.044). However, only in large lesions (&gt; 1.5 cm³) was high approximate OCC (&gt; 10%) associated with lower iPTH levels (adjusted odds ratio for “volume by content” interaction 0.02, 95% CI 0.01–0.52, <i>p</i> = 0.018). Qualitatively similar results were obtained with approximated absolute OCC as the variable of interest.</p> Conclusion <p>In patients with pHPT, a higher approximate OCC was associated with slightly lower calcium concentrations, possibly mediated by a counter regulatory effect on iPTH production, especially in large lesions.</p>