Studies demonstrating calcitonin receptors on Leydig cells have suggested that these cells may be one of the many sites affected by this peptide. To investigate this possibility, the effect of synthetic salmon calcitonin on the TM3 Leydig cell line (derived from immature mouse Leydig cells) and on primary Leydig cell-enriched preparations was examined. Synthetic salmon calcitonin stimulated the conversion of [3H]adenine to [3H]cyclic AMP in TM3 cells. In addition, the hormone stimulated the basal secretion of testosterone in both TM3 cell- and Leydig cell-enriched cultures and potentiated the action of hCG on Leydig cell-enriched cultures. Synthetic salmon calcitonin also increased the concentration of androgen and estrogen receptors in cultured TM3 Leydig cells by 2- and 4-fold, respectively, when added to the culture medium (1 micrograms/ml). The fact that 8-bromo-cyclic AMP decreased both androgen and estrogen receptor concentrations suggested that the effect of calcitonin on sex steroid receptors is not mediated by its effect on cyclic AMP in these cells. The possibility that the action of calcitonin on steroid receptors might be mediated by another messenger such as calcium (Ca2+) was therefore considered. Progressively lowering the concentration of Ca2+ in the culture medium of the cells from 1.5 mM to less than 0.01 mM decreased the concentration of both androgen and estrogen receptors. Returning the Ca2+ concentration to normal levels (1.5 mM) restored steroid receptor levels. Receptor levels were also decreased when the extracellular Ca2+ concentration was lowered to 0.5 mM, and treatment with the Ca2+ ionophore, A23187 (1 microM), restored receptor levels to normal. The calcium channel blocker, verapamil, decreased the androgen receptor concentration but unexpectedly increased the concentration of estrogen receptors. It was concluded that calcitonin stimulates cAMP formation and testosterone secretion, and increases the concentration of sex steroid receptors. These observations provide evidence that the previously demonstrated calcitonin receptors on Leydig cells may be coupled to several biologic responses in this cell type.