To test the protective effect of Towne live attenuated human cytomegalovirus (HCMV) vaccine in normal individuals, we developed a parenteral challenge consisting of a low-passage isolate (Toledo stain) inoculated subcutaneously in graded doses. This challenge virus caused a mild mononucleosis syndrome in seronegative individuals at doses of 10 or 100 pfu. The illness was accompanied by atypical lymphocytosis, raised hepatic enzymes, excretion of HCMV and HCMV-specific immune responses. Naturally seropositive volunteers also developed clinical and laboratory evidence of infection after challenge with 1,000 pfu of Toledo but resisted 10 or 100 pfu. Volunteers who had been vaccinated 1 y earlier also were resistant to disease caused by 10 or 100 pfu of Toledo, although some were asymptomatically infected by the 100 pfu dose. Vaccine-induced immunity to HCMV was as complete as naturally induced immunity when the challenge dose of Toledo was 10 pfu.