Female tilapia, Oreochromis mossambicus, were treated with luteinizing hormone-releasing hormone analogue (LH-RHa) and pimozide (PIM) to stimulate gonadal development and sexual maturation. Twenty-four hours after LH-RHa + PIM or vehicle (controls) injections, ovarian samples were collected for the in vitro incubations. Stage III or Stage IV follicles were treated with either 1) 20 μg tilapia hypophysis plus 50 ng 17-hydroxyprogesterone (17-P), 2) 20 μg hypophysis alone, 3) 50 ng 17-P alone and 4) media alone. Free and conjugated (sulfates and glucuronides) levels of the established teleost oocyte maturation inducing steroids (MIS), i.e. 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) were measured in the incubation media by high performance liquid chromatography. The highest levels of 17,20β-P, in both free and conjugated forms, were measured in media containing Stage IV follicles (up to 7 ng/ml; P < 0.01). Free levels of 17,20β,21-P were detectable only in incubates of Stage III follicles (up to 1 ng/ml). The levels of conjugated 17,20β-P and 17,20β,21-P were higher as sulfates than as glucuronides for all incubates. These results demonstrate that 17,20β-P and 17,20β,21-P are synthesized in vitro by follicles of tilapia and that sulfation is the main route for the metabolism of the C 2 1 -steroids. The high levels of 17,20β-P and 17,20β-P-sulfate in incubates containing Stage IV follicles points at 17,20β-P, rather than 17,20β,21-P, as the most probable MIS in tilapia and suggests a probable pheromone role for free 17,20β-P and 17,20β-P-sulfate in this species.