Calcium is a known intracellular second messenger in many epithelial cells, so its possible role in the secretion of chloride by the rectal gland of the shark was examined using a variety of approaches. The use of ionophores to increase intracellular calcium showed neither stimulation nor inhibition. Calcium channel blockers, to prevent extracellular calcium from entering the cell, showed either nothing or inhibition. The use of calmodulin inhibitors, thus blocking the intracellular mediator of calcium effects, resulted in reduced activity. Blocking the release of calcium from the endoplasmic reticulum caused inhibition, and preventing the reuptake of calcium into the endoplasmic reticulum inhibited oxygen consumption, so reducing or increasing intracellular calcium gave equivocal results. Measurements of intracellular calcium showed that maneuvers that should increase intracellular calcium do so in slices, isolated tubules, or micro-dissected tubules of the rectal gland; thus the level of intracellular calcium responds appropriately to agonists. Elements of calcium-activated pathways such as protein kinase C and diacylglycerol are present in the rectal gland, as may also be the calcium-sensing receptor. Unfortunately, there is not at present clear-cut evidence that calcium participates in the secretion of chloride by the rectal gland.