BACKGROUND We have previously demonstrated that nitric oxide (NO) inhibits steroidogenesis via a cGMP-independent process, by inhibiting P450 aromatase activity in porcine granulosa cells (PGCs) derived from medium-sized (3--5 mm) ovarian follicles (M-PGC). OBJECTIVE To determine whether the NO/NO synthase (NOS) system exerts any significant effects on steroidogenesis in PGCs derived from small follicles (<3 mm) (S-PGC) in comparison with those derived from medium follicles. METHODS PGCs, namely S-PGC and M-PGC, were incubated with the NO donor, NOC18, and a competitive blocker of NOS, N(3)-monomethyl-l-arginine (LNMMA), either alone or in the presence of FSH (200 ng/ml) or hCG (5 IU/ml). RESULTS NOC18 significantly (P<0.01--0.001) suppressed basal (unstimulated) and gonadotropin-stimulated estradiol (E2) release from both S-PGC and M-PGC in a 2-h culture. NOC18 significantly (P<0.01--0.001) decreased basal and gonadotropin-stimulated progesterone release from S-PGC, but not from M-PGC. In addition, NOC18 significantly (P<0.05--0.001) inhibited aromatase activity in S-PGC. LNMMA had a significantly (P<0.01--0.001) stimulatory effect on the basal release of E2 and progesterone from M-PGC; however, it had no significant effect on basal steroidogenesis in S-PGC in a 24-h culture. In the presence of gonadotropin, LNMMA significantly (P<0.01--0.001) stimulated the release of E2 and progesterone from both S- and M-PGC, and this stimulatory effect was weaker in S-PGC than in M-PGC. These results demonstrate that NO inhibits E2 secretion by directly inhibiting the aromatase activity in S-PGC, as in M-PGC. It has been shown that the NO system suppresses the differentiation of S-PGC; however, the extent of suppression decreased with the progression of follicular growth. In addition, the activity of NOS in S-PGC was weaker than that in M-PGC. CONCLUSIONS We strongly suggest that the NO/NOS system in PGC regulates steroidogenesis differently during different phase of follicular development.