We used a PCR method to develop a diagnostic assay for the detection of cytomegalovirus (CMV) DNA in infantile hepatitis, which has been suggested to be associated with CMV infection. CMV DNA was detected in 25 (58.1%) of 43 patients with elevated serum alanine aminotransferase (ALT) levels but no jaundice, and no hepatitis B or C as assessed by conventional PCR. None of the samples from 97 healthy infants tested positive for CMV DNA. We assayed CMV DNA quantitatively in blood using a real-time PCR system that allowed reproducible detection of at least 10 copies of CMV DNA. When 1 microg of DNA from each blood sample was used in this system, a good correlation was obtained between the calculated and measured copy numbers of CMV DNA. This system detected CMV DNA in 29 patients (67.4%) with liver dysfunction. Serial studies in patients with liver dysfunction revealed that CMV DNA copy number decreased, ultimately to below 10, as the ALT levels normalized. In contrast, no CMV DNA copies were detectable by the real-time system in any of the samples from control subjects. These results highlight the usefulness of detecting CMV DNA in the diagnosis of infantile hepatitis and indicate that the real-time quantitative PCR assay may be a valuable tool for monitoring CMV-associated infantile hepatitis.