We have examined the ability of selected hormones and growth factors to suppress the spontaneous onset on apoptotic DNA fragmentation in isolated vitellogenic rainbow trout ovarian follicles cultured in serum‐free conditions. Primary culture of isolated follicles for 24 hr in serum‐free conditions resulted in a 3–5‐fold increase in the amount of fragmented DNA as compared to non‐cultured controls, measured by radioactive 3′end‐labeling. Culture in medium containing salmon gonadotropin (SG‐G100; 1, 5 μg/ml) suppressed the spontaneous onset of DNA fragmentation in dose‐dependent fashion. Culture with 1 ng/ml 17β‐estradiol, or 100 ng/ml epidermal growth factor also suppressed the spontaneous onset of apoptosis, whereas culture with higher concentrations of 17β‐estradiol (10 and 100 ng/ml), insulin‐like growth factor I (IGF‐I; 1, 10, and 100 ng/ml), or 8‐bromo‐cAMP (0.1, 1, and 5 mM) was ineffective in suppressing apoptosis. Apoptosis was confirmed as the mode of cell death through positive identification of nuclear morphological characteristics associated with apoptosis, and positive staining for fragmented DNA using in situ end‐labeling (TUNEL); apoptotic cells identified in situ were almost exclusively localized to the thecal/epithelial region of the follicle. In summary, this study shows that vitellogenic ovarian follicles are susceptible to apoptosis and that both endocrine and locally‐derived growth factors may play a role as cell survival factors by preventing apoptosis. The study also suggests that rainbow trout differ markedly from mammals both in terms of the cell types susceptible to apoptosis and the responsiveness to specific growth factors in terms of inhibiting apoptosis. Mol. Reprod. Dev. 61:511–518, 2002. © 2002 Wiley‐Liss, Inc.