IFN-gamma and/or LPS induced nitrite production and inhibition of Chlamydia trachomatis (CT) replication in the murine macrophage cell line, RAW264.7. Linear regression analysis demonstrated a strong correlation between nitrite production and inhibition of CT replication (correlation coefficients: -0.93, P < 0.001). L-NMMA specifically inhibited nitrite production and restored CT replication (55-71%). Inducible nitric oxide synthase (iNOS) mRNA was analyzed by Northern and dot blot hybridization with an iNOS cDNA probe. A strong correlation between iNOS mRNA expression and inhibition of CT replication also was observed (correlation coefficient: -0.97, P < 0.05). Furthermore, anti-TNF-alpha antibody, which completely neutralized biological activity of the secreted TNF-alpha, neither inhibited nitrite production nor restored CT replication in the LPS- and/or IFN-gamma-treated RAW264.7 cells. In mouse peritoneal macrophages treated with IFN-gamma, both L-NMMA and anti-TNF-alpha antibody inhibited nitrite production and restored CT replication. However, L-NMMA and the antibody had no effect upon nitrite production and CT inhibition in LPS-treated peritoneal macrophages. These data indicate that NO production is one mechanism for inhibition of CT replication in IFN-gamma-activated murine macrophages.