The role of Concanavalin A (ConA), Phytohemagglutinin (PHA) and Wheat germ agglutinin (WGA) in the activation of murine peritoneal macrophages particularly with reference to production and regulation of nitric oxide (NO) has been investigated. Macrophages on treatment with ConA and PHA showed significantly enhanced production of NO, which was dose and time dependent. On the other hand macrophages treated with WGA did not produce NO. L- N-monomethyal- l-arginine (L-NMMA), an inhibitor of NOS inhibited the ConA and PHA induced NO production. ConA and PHA treatment of macrophages induced transcription of iNOS gene and the enhanced expression of iNOS protein. Pharmacological inhibitors of PI3 kinase—Wortmannin, tyrosine kinase—Genestein, protein kinase C—H-7 and p42/44—PD98059 inhibited the ConA and PHA induced production of NO and p38 MAP kinase inhibitor SB202190 inhibited NO production only in ConA treated macrophage, while Gαi protein inhibitor—PTX and JNK inhibitor—SP600125 inhibited NO production in PHA treated macrophages. Tyrophostin (AG490), an inhibitor of JAK2 and TMB-8, an intracellular calcium immobilizing agent also inhibited the ConA and PHA induced NO production, suggesting the involvement of JAK-STAT pathway and calcium. The data also provides the relative measure and importance of different key signaling molecules in the regulation of NO production by macrophages on activation.