Mycobacterium avium complex (MAC) can induce upregulation of HIV. To investigate the underlying mechanisms, the effect of MAC-induced cytokines on HIV replication was first studied. Semiquantitative RT-PCR, followed by Northern blot analysis, revealed that mRNA encoding IL-6 and TNF-alpha was induced by MAC. However, production of these cytokines was undetectable and the addition of anti-cytokine antibodies to coinfected cells could only minimally block the MAC effect on HIV. Infection of U38 cells with MAC resulted in enhancement of HIV-1 LTR-CAT transcription. In addition, transient transfection of U937 cells with full-length wild-type as well as NF-kappaB-binding site-deleted mutant HIV-1 LTR-CAT constructs revealed that MAC-induced HIV-LTR CAT is NF-kappaB dependent. These findings, together with our previous work, indicate that MAC-induced cytokine expression increases the formation of NF-kappaB, which in turn enhances HIV-1 LTR-CAT transcription. However, additional factor(s) yet to be elucidated may play a more significant role in MAC-mediated HIV-upregulation.