Parameters for plaque formation by rubella vaccine strains licensed in Japan were studied. Formation of clear and large plaques on RK13 cells depends on several essential parameters. Plaques differed in morphology among five vaccine strains and the distinctiveness of the plaques was affected by pH of the agar overlay medium during incubation at 35 degrees C. Plaques became progressively larger in size as the concentration of sodium bicarbonate in the agar overlay medium increased from 0.04% to 0.15%, but the contrast of plaques to the background cells decreased markedly. The most distinct plaques of all vaccine strains were formed in the agar overlay medium containing 0.07% of sodium bicarbonate, i.e., pH 6.83, incubated in a humidified atmosphere containing 5% CO2. The number of plaques formed by vaccine strains decreased at 37 degrees C. Vaccine strains other than MEQ11 and TCRB19 formed larger and more contrasted plaques with sharp outline at 35 degrees C than at 32 degrees C. MEQ11 and TCRB19 strains yielded higher infective virus titres at 32 degrees C, but they formed distinct plaques at 35 degrees C and 32 degrees C. For the plaque test, the inoculum volume was another critical factor for obtaining an approximate titre that reflected the absolute titre of the sample. A volume of 0.1 ml was feasible for a well with a diameter of 35 mm.