Despite the eradication of smallpox as a naturally occurring disease, concern persists over its potential use as a bioterrorist agent. The development of a new-generation smallpox vaccine represents an important contribution to a cogent biodefense strategy. We conducted a phase 2 randomized, double-blind, controlled trial at four sites in the United States to determine whether a clonal smallpox vaccine manufactured in cell culture, ACAM2000, is equivalent to the standard calf-lymph vaccine, Dryvax, in terms of cutaneous response rate, antibody responses and safety. Subjects received either Dryvax or one of four dose levels of ACAM2000 administered percutaneously using a bifurcated needle. All subjects in the highest ACAM2000 dose group and the Dryvax group experienced a successful vaccination. Dilution doses of ACAM2000 were associated with success rates below the 90% threshold established for efficacy. There were no differences in the proportion of subjects who developed neutralizing antibody: 94% in the highest ACAM2000 dose group (95% CI, 84-99) and 96% in the Dryvax group (95% CI, 86-100). No significant differences were seen between the effective ACAM2000 and Dryvax groups regarding the occurrence of adverse events. One subject who received ACAM2000 developed myopericarditis. In healthy, primary vaccines ACAM2000 has a similar vaccination success rate, antibody response, and safety profile to Dryvax.