The generation of nitric oxide (NO) by activated macrophages is believed to control mycobacterial infection in the murine system. In this study we examined the effect of Mycobacterium bovis BCG infection on the L-arginine-dependent NO pathway in J774.1 murine macrophages. We have confirmed previous results by demonstrating that stimulation of J774.1 with lipopolysaccharide (LPS) and gamma interferon (IFN-) results in an increase in the uptake of ³H-labeled L-arginine and a concomitant increase in the production of NO. We have also shown that BCG can mimic LPS treatment, leading to enhanced L-[³H]arginine uptake by IFN--stimulated macrophages. Lipoarabinomannan, a component of the BCG cell wall that is structurally similar to LPS, is not responsible for the uptake stimulation in IFN- stimulated macrophages. Although we demonstrated that there was a 2.5-fold increase in NO production by macrophages 4 h after LPS-IFN- stimulation, BCG infection (with or without IFN- stimulation) did not lead to the production of NO by the macrophages by 4 h postinfection. At 24 h postinfection, the infected macrophages that were stimulated with IFN- produced amounts of NO similar to those of macrophages stimulated with LPS-IFN-. This suggests that there are multiple regulatory pathways involved in the production of NO. Finally, our data suggest that increased expression of the arginine permease, MCAT2B, after 4 h of LPS-IFN- treatment or BCG infection-IFN- treatment is not sufficient to account for the increases in L-[³H]arginine uptake detected. This suggests that the activity of the L-arginine transporter(s) is also altered in response to macrophage activation.