Proinflammatory cytokines, including tumor necrosis factor (TNF)alpha, have been recognized as important physiopathogenetic factors in the initiation and continuation of inflammatory cardiomyopathies. Experimental and preliminary human studies have demonstrated that TNFalpha plays a crucial role in enteroviral-induced myocarditis. In this study, we investigated the expression of TNFalpha and both its receptors (TNFRI and TNFRII) in both viral and nonviral myocarditis. Myocardial expression of TNFalpha was then correlated with different clinical and pathologic findings. TNFalpha expression was investigated in endomyocardial biopsies obtained from 38 patients with myocarditis and from eight control subjects by using reverse transcriptase-polymerase chain reaction (PCR) and immunohistochemistry. Viral etiology was diagnosed by PCR in 20 cases: enterovirus in seven, Epstein-Barr virus in four, hepatitis C virus in three, adenovirus in two, influenza virus in two, cytomegalovirus in one, and double infection adenovirus and enterovirus in one. Immunohistochemistry was also used to analyze both TNFalpha receptors (RI and RII). A semiquantitative analysis was employed (score 0-3) for necrosis, inflammation, fibrosis and immunohistochemical findings. TNFalpha mRNA and TNFalpha protein were significantly more present in viral myocarditis than in nonviral myocarditis (16/20 vs 3/18, P=0.001). Remarkable immunostaining was observed for both receptors, particularly TNFRI. Histological analysis revealed that myocardial necrosis (mean score 1.89 vs 1.15, P=0.01) and cellular infiltration (mean score 2.26 vs 1.78, P=0.05) were more prominent in TNFalpha-positive cases. Among TNFalpha-positive cases, the greater TNFalpha mRNAs, the more impaired was cardiac function. Our findings suggest that the expression of TNFalpha may play an important role in the pathogenesis of viral myocarditis of any etiology and may influence the severity of cardiac dysfunction. Cytokine effects are more strictly linked to overexpression of TNFRI.