Cytomegalovirus (CMV) hepatitis is a common and serious complication of orthotopic liver transplantation. Immunohistochemical studies are the most sensitive methods of diagnosis. We compared immunoperoxidase staining with monoclonal antibodies to CMV immediate early and early antigens with routine hematoxylin-eosin stain. Eleven of 140 liver allograft recipients at our institution had CMV hepatitis identified by hematoxylin-eosin stain on biopsy specimens. We stained serial sections of all previous biopsy specimens and one post-ganciclovir biopsy specimen (when available) from each of these patients. One or both monoclonal antibodies confirmed the original hematoxylin-eosin stain diagnoses. Cytomegalovirus was detected in earlier, hematoxylin-eosin stain-negative biopsy specimens in seven of 11 patients. Detection of immediate early antigen often preceded that of early antigen. Earlier biopsy specimens demonstrated less positive staining, which become more extensive closer in time to the hematoxylin-eosin stain-positive biopsy specimens. Sinusoidal cells became positive earlier than hepatocytes. In one patient occult CMV antigens persisted in biopsy specimens following ganciclovir treatment. We conclude that (1) immunohistochemical staining for CMV antigens can result in earlier detection of viral infection, which may lead to earlier, more effective treatment; (2) CMV infection and antigen expression is focal, requiring extensive examination for diagnosis; (3) extent of occult infection may indicate the extent of active infection in the organ as a whole; (4) most CMV hepatitis begins with infection of sinusoidal lining cells as a result of hematogenous spread from within the allograft or from systemic viremia; and (5) posttreatment biopsy specimens may be more sensitive than resolution of serum liver enzyme abnormalities in evaluating the success of ganciclovir therapy.