Cytosolic free Ca2+ ion concentrations were ([Ca2+]i) measured in single swine granulosa cells using the Ca2(+)-sensitive fluorescent indicator dye fura-2 and digital imaging videomicroscopy with high spatial and temporal resolution. Ovine FSH (oFSH) elicited specific [Ca2+]i increases, which reached their highest value within 5 min of the onset of stimulation and were sustained for 10-15 min. [Ca2+]i usually returned to prestimulated levels and cells regained full responsiveness to a second exposure to oFSH within 20 min. The effect of FSH was not blocked by pretreatment with pertussis toxin. Forskolin and 8-bromo-cAMP were able to mimic similar increases in [Ca2+]i in the presence or absence of extracellular Ca2+. In contrast, the oFSH-induced [Ca2+]i rises were abolished in the absence of extracellular Ca2+. The Ca2+ channel blocker verapamil completely abolished the oFSH-induced [Ca2+]i rise, but not the 8-bromo-cAMP- or the forskolin-induced [Ca2+]i rise. In summary, we have demonstrated that [Ca2+]i is regulated by oFSH in single swine granulosa cells. We propose that the transducing pathway for this FSH effect might not involve cAMP, and that whichever second messenger is responsible for the Ca2+ signal generation acts upon plasma membrane Ca2+ channels.