Dilution of viruses, which were previously treated with 10 micrograms acetyltrypsin/ml, in MEM supplemented with 2.5% fetal bovine serum caused little damage of cell monolayers resulting in the formation of discrete plaques. When agar and acetyltrypsin were added to the overlay medium at the concentrations of 0.8% and 5 micrograms/ml, respectively, clear plaques were induced. Linear relationships were obtained between virus concentration and the number of plaques, thus the procedure described in this report might be used for reliable plaque assay of rotaviruses. Moreover, these conditions for plaque formation were successful for estimating isolated viruses, as well as standard strains.