Regulation of steroidogenesis by luteinizing hormone is mediated by cAMP and calcium. We have investigated changes in cytosolic free calcium ([Ca2+]i) in Leydig cells by using Fura-2 as the fluorescent calcium indicator. Purified Leydig cells were plated on polylysine coated glass coverslips, cultured for 24 h in DMEM/F12 and loaded with Fura-2 at 37 degrees C. [Ca2+]i measurements were made fluorometrically by placing coverslips into 3 ml cuvettes with PBS+calcium. Addition of hCG increased [Ca2+]i gradually after a lag of about 2-3 min and plateaued by 5-6 min. The plateau level was sustained for at least 15 min. Absence of external Ca2+ in the medium or presence of diltiazem or nicardipine or cobalt chloride abolished the rise. Addition of BAY K 8644 or KCl caused a small but significant increase of [Ca2+]i. 8-Br-cAMP, forskolin or cholera toxin produced a gradual rise in [Ca2+]i that plateaued after 5-6 min similar to that observed with hCG. The action of hCG was inhibited by protein kinase A inhibitor (20-residues peptide) but not by protein kinase C inhibitor (staurosporine). We conclude that binding of hCG to its receptors would transmit the signal through G proteins to adenylyl cyclase to increase cAMP which would increase Ca2+ influx into cytosol across plasma membrane Ca2+ channels. Therefore, it appears that the primary action of hCG is to increase cytosolic cAMP which would then regulate [Ca2+]i as well as steroidogenesis.