Pediatric myocarditis is a serious disease resulting in significant morbidity and mortality. Tracheal aspirate (TA) has been demonstrated to be a sensitive diagnostic tool to detect viral agents responsible for respiratory disorders and myocardial dysfunction. Tumor necrosis factor alpha (TNFalpha) is thought to play an important role in the pathogenesis of these disorders. The aim of the present study was to investigate the presence of different viruses and the expression of TNFalpha in children with clinical suspicion of myocarditis. Forty-five TAs from children (20 males/25 females, mean age 4.4+/-5.0 y) with myocardial dysfunction and respiratory symptoms were analyzed for detection of viral genomes by using molecular techniques. In 10 cases endomyocardial biopsy was also performed due to a severe and rapid progression of heart failure. TNFalpha mRNAs of TA and TNFalpha protein plasma levels were quantified. Viral etiology was detected in 25/45 (56%) cases: the most frequent etiology was enterovirus (19 cases, 59%). Polymerase chain reaction viral concordance was found in TA and endomyocardial biopsy. TNFalpha mRNA and TNFalpha serum levels were significantly more expressed in viral cases than nonviral cases (1.26+/-0.76 vs. 0.56+/-0.76, P=0.001). More impaired cardiac function (particularly ejection fraction) was detected in viral positive than in viral negative cases (39.91+/-20.09 vs. 55.61+/-20.36, P=0.04). TA seems to be an excellent tool for viral investigation in pediatric patients with suspicion of myocarditis. The analysis of TNFalpha in TA may represent an important marker to better define patient status.