A protein immunoblot (western blot) assay was developed for detection of IgG and IgM antibodies to cytomegalovirus (CMV) in banked serum and was used to study the epidemiology of infection in a breeding population of rhesus macaques. All juveniles and adults were antibody-positive to CMV. Repeated samples from 1990-born infants (n = 28) over a 12-month period allowed retrospective assessment of CMV seroconversion. Most infants (23 of 25 seroconversions) were apparently exposed to CMV during their first year of life. The IgM testing permitted identification of seroconversion time points in eight infants. Risk factors that significantly affected seroconversion probabilities in the study population were not found. Cytomegalovirus-free rhesus macaques are potentially valuable for viral teratology and pathogenesis research projects; however, present epidemiologic findings suggest that establishment of CMV-free colonies will be difficult in conventional settings.