500 sera representing healthy blood donors and a random representation of the U.S. population collected 10 years ago were screened by ELISA for antibody reactivity with purified, disrupted HBLV virions. In each group, the ELISA results were normally distributed with no evidence of bimodality. All sera were subsequently retested after preincubation of each with well-characterized preparations of disrupted HSB-2 cells or HBLV-infected HSB-2 cells. Sera showing significant levels of HBLV-specific neutralization (50% or more) were found in Minneapolis, Kansas City, and in a random population survey (81, 88 and 97% of donors, respectively). Mean ELISA test values were the same for all groups and for males and females within the same group. Sera from these normal donors reacted preferentially with viral antigens of 120 and 58 kDa by Western blot. In a hospital-based prevalence study, frequent IgM and IgG seroconversions were apparent among infants less than 1 year old, and mean ELISA test values reached the adult level before school age. Antigen preparations used in blocking experiments showed no competitive cross-reactivity with antisera against EBV, CMV, HSV, VZV, HIV, or adenovirus type 2 at levels which reduced antibody binding to HBLV by more than 90%. Antibody cross-reactivities towards HBLV and other human herpesviruses were assessed by cross-correlation of viral antibody titers against all of the viruses and by cross-absorptions of antisera against the other viruses with HBLV. In these experiments no antibody cross-reactivity between HBLV and other human herpesviruses were detected. The significance of these findings with respect to health/disease status is presently unknown. Further seroepidemiologic studies of quantitative levels of HBLV antibody reactivity to measure the age of primary infection and progressive changes in healthy and selected disease populations are needed to determine the risk of disease associated with HBLV infection.