The induction of interleukin-6 (IL-6) using combined proinflammatory agents (LPS/IFN-γ or TNF-α/ IFN-γ) was studied in relation to p38 mitogen-activated protein kinase (MAPK) and NF-κB transcriptional factor in primary neonatal cardiomyocytes. When added to cultures of cardiomyocytes, the combined agents (LPS/IFN-γ or TNF-α/IFN-γ) had stimulatory effect on the production of IL-6 and the elevation was significantly reduced by SB203580, a specific p38 MAPK inhibitor. SB203580 inhibited protein production and gene expression of IL-6 in a concentration-dependent manner. In this study, IFN-γ enhancement of TNF-α-induced NF-κB binding affinity as well as p38 MAP kinase activation was observed. However, a specific inhibitor of p38 MAPK, SB203580, had no effect on TNF-α/IFN-γ or LPS/IFN-γ-induced NF-κB activation. This study strongly suggests that these pathways about TNF-α/ IFN-γ or LPS/IFN-γ-activated IL-6 release can be primarily dissociated in primary neonatal cardiomyocytes.