It was established that microfocus assay of the variola virus in HeLa cell cultures on Microtiter plates is a suitable method for titrating the virus in numerous specimens of small volume. The variola foci were formed within 48 hours of inoculation, and the standard error for the number of foci per well did not exceed 15%, with 96 flat-bottomed wells (6 mm diameter) containing 0.1 ml per well of the culture media on a plastic plate replacing 96 Petri dishes in the standard assay procedures. Microfocus assay was successfully applied to the quantification of neutralizing antibody against variola and vaccinia viruses, with a reproducibility equal to that of the standard focus reduction method on glass Petri dishes. Applications of this technique to the assay of field specimens and to the laboratory differentiation of variola viruses are discussed.