Synthesis of nitrite (NO2-) and nitrate (NO3-) was studied in the macrophage cell lines RAW 264.7, WEHI-3, PU5-1.8, J774A.1, and P388D1 and compared to the synthesis by thioglycolate-elicited peritoneal macrophages from C3H/He and C3H/HeJ mice. Treatment with Escherichia coli lipopolysaccharide (LPS) induced NO2-/NO3- synthesis by all the cell lines except P388D1, which remained unresponsive at the highest LPS concentration (50 micrograms/ml). Recombinant murine gamma-interferon induced NO2-/NO3- synthesis in only two cell lines (PU5-1.8 and RAW 264.7), although it activated synthesis by C3H/He and C3H/HeJ macrophages. Dual signal treatments consisting of lymphokines or gamma-interferon plus LPS stimulated NO2-/NO3- synthesis by all five cell lines and each line showed enhanced synthesis as compared to that induced by any single stimulus. Heat-killed Bacillus Calmette-Guérin and purified mycobacterial protein derivative stimulated NO2-/NO3- synthesis in three of five cell lines, while dextran sulfate, zymosan, and the synthetic adjuvant muramyl dipeptide were ineffective. Nitrite represented 50-75% of the total NO2-/NO3- produced in all cases. The kinetics of LPS-induced NO2-/NO3- synthesis in J774A.1 and C3H/He macrophages were identical; a 6-h lag phase was followed by a 24- to 48-h period in which NO2- and NO3- were in a ratio of approximately 3:2 at all time points.