Development of a vaccine for prevention of congenital cytomegalovirus (CMV) disease is a priority. This study evaluated a "prime-boost" strategy by comparing the safety and immunogenieity of 3 doses of subunit CMV glycoprotein B (gB) vaccine plus MF59 (a squalene-in-water emulsion), 2 doses of a eanarypox recombinant vaccine expressing CMVgB (ALVAC-CMVgB) followed by 2 doses of the subunit gB vaccine, 3 doses of both vaccines administered concomitantly, and placebo in 105 healthy, CMV-seronegative adults. Systemic adverse events were rare, but local reactions were common in all groups. After the first subunit vaccination, neutralizing antibody titers in the prime-boost group were comparable to those in subjects receiving 2 subunit vaccinations, indicating a priming effect of ALVAC-CMVgB. However, after the final dose, antibody and cell-mediated immune responses were not significantly different among the groups. All 3 vaccine regimens induced high-titer antibody and lymphoproliferative responses, but no benefit for priming or simultaneous vaccination was detected.