A total of 879 paraffin-embedded endomyocardial biopsy specimens from 69 heart transplant recipients were studied. In 30 biopsy specimens, the presence of human cytomegalovirus was investigated by routine histologic and immunohistochemical evaluation, in situ hybridization, and polymerase chain reaction. These 30 biopsies were performed in seven patients with clinical human cytomegalovirus infection (four primary and three recurrent infections) and in eight patients with asymptomatic human cytomegalovirus recurrent infection. These endomyocardial biopsy specimens showed grade 0 (n = 9), 1A (n = 12), 1B (n = 7), or 2 (n = 2) acute rejection. No myocarditis with human cytomegalovirus-like inclusion bodies was observed by routine histologic evaluation. Human cytomegalovirus DNA or antigens were not shown by in situ hybridization or by immunohistochemical evaluation, respectively. Viral DNA was detected by polymerase chain reaction in two grade 1A endomyocardial biopsy specimens from two patients with systemic human cytomegalovirus primary infection. These two biopsy specimens were shown to be positive by polymerase chain reaction at the time of the acute phase of the infection as shown by laboratory findings. Therefore cytomegalovirus DNA detected by polymerase chain reaction could result from viral carriers, that is, leukocytes of rejection-related infiltrates or within intramyocardial vessels as a result of a more aggressive expression of the systemic infection in seronegative recipients with cytomegalovirus seropositive donors. Polymerase chain reaction is the most sensitive method for viral DNA detection on paraffin-embedded biopsy specimens, but a multitechnologic approach, including routine histologic evaluation, is required for a proper diagnosis of human cytomegalovirus myocardial infection.