Infection with vesicular stomatitis virus (VSV) of human diploid cells preinfected with the AD-169 strain of human cytomegalovirus (CMV) resulted in the formation of a VSV (CMV) pseudotype. Its formation was favored by increasing the bicarbonate content in doubly-infected cultures. The pseudotype was capable of infecting not only human but also rabbit cells. Pseudotype particles formed after infection with the tl 17 mutant of VSV, which carries a thermolabile lesion in its neutralization antigen, were more stable at 45° than the original tl 17 virus. The pseudotype was used in the neutralization test with human sera. All sera positive for CMV antibody in the complement-fixation (CF) test were also reactive in the neutralization test. In addition, numerous sera negative for CMV antibody in the CF test neutralized the pseudotype.