Leydig-cell suspensions, prepared from rat testes, were incubated with different amounts of Ca 2+ with and without added luteinizing hormone. The basal testosterone production in the absence of luteinizing hormone was unaffected by the Ca 2+ concentration in the incubation medium. The luteinizing hormone-stimulated testosterone production, however, was progressively decreased in the absence of Ca 2+ to one-third of that with 2.50 mM-Ca 2+ . This decrease in luteinizing hormone-stimulated testosterone production was independent of the different concentrations of luteinizing hormone (0-10μg/ml) used and could be restored by the addition of Ca 2+ to the incubation medium. The restoration of the stimulation was achieved within 30 min after the addition of Ca 2+ to the medium. Activation of cyclic AMP-dependent protein kinase by luteinizing hormone was not decreased by omission of Ca 2+ from the incubation medium, suggesting that Ca 2+ may be involved in steroidogenesis at a stage beyond the luteinizing hormone receptor-adenylate cyclase-protein kinase system.