Human immunodeficiency virus (HIV) infection is associated with a surprisingly high frequency of myocardial dysfunction. Potential mechanisms include direct effects of HIV, indirect effects mediated by cytokines, or a combination. We have previously reported that interleukin-1 (IL-1) (500 U/ml) alone induced nitric oxide (NO) production by neonatal rat cardiac myocytes (CM). Effects of the HIV-1 envelope, glycoprotein120 (gp120), on inducible NO synthase (iNOS) in CM have not been previously reported. Unlike IL-1, recombinant HIV-gp120 (1 µg/ml) alone failed to enhance NO production in CM (0.5 ± 0.4 vs. 0.4 ± 0.5 µmol/1.25 × 10⁵ cells/48 h, gp120 vs. control, respectively; n = 12, P = not significant). However, the addition of gp120 to IL-1 significantly enhanced iNOS mRNA expression (70 ± 1.5 vs. 26 ± 2.4 optical units, IL-1 + gp120 vs. IL-1, respectively; n = 3), iNOS protein synthesis (42 ± 1.4 vs. 18 ± 0.8 optical units, IL-1 + gp120 vs. IL-1, respectively; n = 3), and NO production (NO₂) (6.6 ± 0.6 vs. 4.1 ± 0.8 µmol/1.25 × 10⁵ cells/48 h, IL-1 + gp120 vs. IL-1, respectively; n = 12, P  0.5). HIV-gp120 enhancement of IL-1-induced NO₂ production was blocked by 10 µM of SB-203580 (SB), a selective p38 protein kinase inhibitor (3.6 ± 0.2 vs. 6.6 ± 0.6 µmol/1.25 × 10⁵ cells/48 h, IL-1 + gp120 + SB vs. IL-1 + gp120, respectively; n = 12, P  0.5). HIV-gp120-enhanced p38 protein kinase activity was associated with an increase in IL-1-stimulated NF-B activity (184 ± 12.7 vs. 92 ± 10.7 optical units, IL-1 + gp120 vs. IL-1, respectively; n = 3). None of these effects was seen with another recombinant HIV-1 protein, Tat. Thus HIV-gp120 enhancement of IL-1-induced NO production is associated with p38-mediated activation of NF-B. Direct effects of HIV-gp120 on CM may provide a previously unrecognized mechanism contributing to HIV cardiomyopathy.