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Regulation of steroidogenesis by luteinizing hormone is mediated by cAMP and calcium. We have investigated changes in cytosolic free calcium ([Ca 2+] 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° C.[Ca 2+] i measurements were made fluorometrically by placing coverslips into 3 ml cuvettes with PBS+ calcium. Addition of hCG increased [Ca 2+] 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 Ca 2+ 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 [Ca 2+] i. 8-Br-cAMP, forskolin or cholera toxin produced a gradual rise in [Ca 2+] i that plateaued after 5–6 …