The capacity of granulosa and thecal cells to produce PGF [prostaglandin F] in tissue culture was compared, and the effects on this capacity of exogenous gonadotropin, arachidonic acid and estradiol were observed in rabbits. The mean concentration of PGF in granulosa cell cultures was increased significantly by addition of arachidonic acid or estradiol but not with LH/FSH [luteinizing hormone/follicle-stimulating hormone]. On a per follicle basis, the granulosa generally produced more PGF than the thecal tissue, and in the cultures treated with arachidonic acid the difference between the tissues was highly significant (P L 0.01). Granulosa cells and thecal tissue from preovulatory follicles of the rabbit produce PGF in tissue culture, and the increment in PGF produced after addition of arachidonic acid suggests the presence of an active PG synthetase system. Granulosa cells generally produced more PGF than the theca, even allowing for possible contamination of thecal cultures with granulosa cells. Granulosa cells may be a major source of the PG, the production of which could be steroid (estrogen)-mediated.