This study was conducted to determine whether endothelial‐derived nitric oxide synthase (eNOS) affects meiotic maturation of mouse oocytes in vitro. Cumulus‐oocyte complexes (COC) were isolated from ovarian follicles of 27‐day‐old PMSG‐primed wildtype (WT), and eNOS‐knockout (eNOS‐KO) females, and cultured in drops of medium under oil at 37°C for 16–18 hr. Experiment 1 was carried out to determine effects of eNOS deficiency on the ability of COC to mature in vitro. To determine whether acute synthesis of nitric oxide (NO) was required for oocyte maturation, COC collected from WT mice were cultured in medium without (control) or with different doses of N^ω‐nitro‐L‐arginine methyl ester (L‐NAME), an inhibitor of NOS (exp. 2). To assess effects of NO deficiency on the kinetics of germinal vesicle breakdown (GVBD), COC from WT and eNOS‐KO females were observed for 3.5 hr. COC from WT females were also incubated in medium without or with L‐NAME (exp. 3 and 4). After the culture period, cumulus cells were removed, and oocytes were counted and classified as metaphase II (M II), metaphase I (M I) or showing atypical (degenerative) morphology. To determine viability and nuclear morphology of oocytes, they were stained with fluorescein diacetate or 4,6‐diamidine‐2′‐phenylindole dihydrochloride, respectively. There were no differences in body weights but ovarian weights were lower in eNOS‐KO mice compared with WT mice (P < 0.05). Ovaries from eNOS‐KO mice contained fewer COC collected relative to WT mice (P < 0.01). Maturation of COC from eNOS‐KO mice or WT oocytes treated with L‐NAME resulted in a lower percentage of oocytes at M II stage (P < 0.01 and P < 0.05, respectively) and a higher percentage of oocytes at M I or atypical stages compared with those from WT (P < 0.01 and P < 0.05, respectively). Many oocytes that showed either an arrest in M I stage or abnormal morphology were not viable. Several oocytes in M II stage demonstrated abnormalities in distribution of maternal chromosomes. Our data demonstrate that eNOS‐derived NO is a key modulator of oocyte meiotic maturation in vitro. These results support our previous observations in vivo and indicate that eNOS/NO has independent functions in both oocyte maturation and follicular/oocyte development. Mol. Reprod. Dev. 55:412–421, 2000. © 2000 Wiley‐Liss, Inc.