A protocol for the incubation of Centropristis striata ovarian follicles in vitro from females that spawn repeatedly under laboratory conditions was developed. Isolated follicles underwent maturation after 15–24 h of incubation at 25 °C in 75% Leibovitz L-15 medium, containing relatively low doses of human chorionic gonadotropin (hCG) or homologous pituitary extract (50% response with approximately 2 IU/ml and 0.02 pituitary equivalents/ml, respectively). During maturation in vitro, follicles underwent a remarkable volume increase (about 340%) that was correlated with proteolysis of primarily the largest yolk protein in the oocyte. The maturation response of follicles to hCG was size-dependent within a range from 400 to 600 m in diameter, with a maximum response displayed by 530–600 m follicles. The response of these follicles to hCG was not inhibited within a range of incubation temperatures from 15 to 28 °C, but the length of time required for the follicles to achieve 50% maturation was negatively correlated with increasing temperatures (r2 = 0.97; p ≤ 0.01). However, the largest follicles (530–600 μm) did not undergo maturation in response to high doses (1 μg/ml) of the major maturation-inducing steroids (MISs) known in teleosts, 17.20-dihydroxy-4-pregnen-3-one (17.20β) and 17.20β.21-trihydroxy-4-pregnen-3-one (20βS), even after a previous incubation period of follicles with 10 IU/ml hCG for 1.3 or 6 h. These observations indicate that C. striata follicles are very sensitive to increasing doses of gonadotropin, and that their responsiveness to this hormone is highly size-dependent within a very narrow range. The preliminary results also suggest that 17.20β and 20βS are not the MIS in this species.