In previous work it was shown that the immune cytokine interferon-gamma (IFN-gamma) inhibits hormone secretion in anterior pituitary (AP) cell cultures, an action most likely mediated by folliculostellate (FS) cells. In the present study, we wanted to investigate whether nitric oxide (NO) is involved in this inhibitory action of IFN-gamma. NO synthase (NOS) inhibitors with affinity for the inducible (iNOS) and the constitutive (cNOS) isoform such as N(G)-monomethyl-L-arginine (L-NMMA) and S-methyl-L-thiocitrulline (SMLT) dose-dependently blocked the inhibitory action of IFN-gamma on GHRH-stimulated GH secretion, and partially reversed the inhibitory effect on basal prolactin (PRL) release. In the absence of IFN-gamma these inhibitors significantly augmented basal PRL release and slightly enhanced GHRH-stimulated GH release. L-N6-(1-iminoethyl)lysine (L-NIL), a NOS inhibitor with preferential affinity for iNOS, abrogated the IFN-gamma effect on GHRH-stimulated GH secretion and partially reversed IFN-gamma inhibition of PRL release. However, L-NIL did not exert a stimulatory effect on basal PRL and GHRH-stimulated GH release by its own. 2,4-diamino-6-hydroxypyrimidine (DAHP), a NOS inhibitor by interfering with tetrahydrobiopterin (BH4) cofactor availability, showed the same activity profile as L-NIL. NOS inhibitors blocked or reduced the production of NO as detected by measuring nitrite (NO2-) levels in AP cell cultures and cGMP levels in the NO-reporter cell line RFL-6. The NOS inhibiting action of L-NMMA was confirmed by competition experiments with the natural NOS substrate L-arginine. Thus, in culture medium with lower amounts of L-arginine, L-NMMA blocked the IFN-gamma-induced inhibition of GHRH-stimulated GH release at a lower dose. The inhibition of PRL and GH release by IFN-gamma was markedly reduced in L-arginine-depleted medium. The NO donor sodium nitroprusside (SNP) mimicked the inhibitory action of IFN-gamma on GHRH-stimulated GH and basal PRL release. Similarly to IFN-gamma, SNP did not affect basal GH release. As previously reported, inhibition by IFN-gamma occurred only in AP cell populations containing a minimal proportion of FS cells. As studied in different cell populations obtained by unit gravity sedimentation in a serum albumin gradient, L-NMMA reversed the IFN-gamma effect in the same populations enriched in FS cells. Interestingly, in the absence of IFN-gamma L-NMMA strongly stimulated basal PRL release in the population most enriched in FS cells. It is concluded that IFN-gamma through activation of the iNOS pathway probably in FS cells enhances the production of NO and that this effect is responsible for the inhibitory action of IFN-gamma on GHRH-stimulated GH release and partially for the IFN-gamma-induced decrease in basal PRL release. On the other hand, NO, likely produced by cNOS, appears to exert a tonic inhibitory effect on GHRH-stimulated GH and basal PRL release. It seems therefore that low amounts of NO produced constitutively may take charge of subtle physiological adaptations, and higher levels of NO produced by iNOS under the influence of IFN-gamma may attenuate PRL and GH release during emergency conditions of immune and inflammatory reactions.