We have utilized isolated, highly differentiated porcine granulosa cells maintained under chemically defined conditions in vitro to examine directly the relationship between ornithine decarboxylase (ODC) activity and progesterone secretion in ovarian cells. The administration of saturating concentrations of LH, prostaglandin E2, L-epinephrine, or 8-bromo-cAMP each elicited highly significant increases in progesterone production, which correlated closely with corresponding stimulated levels of ODC activity. Highly purified preparations of FSH elicited no enzymatic or steroidogenic response. ODC activity and progesterone production also correlated closely after administration of various inhibitors of protein or RNA synthesis [cycloheximide (10 microgram/ml), actinomycin D (1 microgram/ml), or alpha-amanitin (1 microgram/ml)]. However, in time-course experiments, progesterone secretion increased significantly before the rise of ODC activity. Furthermore, a direct irreversible catalytic inhibitor of ODC activity, DL-alpha-difluoromethylornithine, and three indirect inhibitors of ODC that putatively induce intracellular antizyme, produced profound suppression of enzymatic activity, without altering maximal progesterone production in response to LH. Thus, the current studies close parallelism between hormonal regulation of ODC activity and progesterone production in ovarian cells in vitro. However, our ability to demonstrate apparent dissociation between these two processes suggests that hormonal stimulation of steroidogenesis may not be obligatorily coupled to the rapidly turning-over cytosolic protein ODC.