Fetal infection with human cytomegalovirus (HCMV) is the leading viral cause of brain damage among newborns at birth or later in life. Efforts to screen newborns routinely for shedding of the virus by immunoassay have been hampered by inhibitors in urine, reportedly the host protein beta2-microglobulin (beta 2m). An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of HCMV antigen in which the reactivity was not affected by the presence of beta 2m, but nevertheless inhibition was observed when urine samples with high levels of virus were tested. The presence of antibodies to HCMV was demonstrated in these urine samples by antibody ELISA and immunoblot using the major antigenic protein of HCMV (pp150) expressed in Escherichia coli; this offers an alternative explanation for the inhibition in ELISA. The presence of HCMV antibodies correlated significantly with congenital HCMV infection (as detected by tissue culture isolation of virus from urine samples of newborns), especially with asymptomatic cases (sensitivity 70%; specificity 94%). The data indicate a local (renal) immune response to HCMV in congenitally infected children, which may have future diagnostic applications.