Persistence of neutralizing antibodies after revaccination against smallpox was studied. Single serum samples from 140 revaccinated donors were tested for neutralizing antibodies using the plaque reduction assay. The donors, aged 21-49 years at sampling, had been vaccinated in infancy and revaccinated at 8 and 18 years; they formed seven groups of 20 men each, revaccinated about 3, 5, 10, 15, 20, 25, and 30 years before sampling. The differences in mean titer among groups were insignificant (P greater than .01). The titer significantly decreased during the first 3 years after the revaccination but remained stable for at least 30 years thereafter (geometric mean titer, 10.5; 95% confidence interval, 6.8-16.4). The results suggest that there is probably no need for routine revaccinations beyond the primary and two revaccinations; nevertheless, persons at high risk should be revaccinated regardless of their vaccination status.