We have previously found that upon passaging type 3 oral poliovirus vaccine (OPV) in cell cultures the proportion of revertants at nucleotide 472 rapidly increases [Chumakov et al.: Proceedings of the National Academy of Sciences of the United States of America 88:199-203 1991]. Systematic study on the accumulation of these revertants showed that it was dependent on the multiplicity of infection and the temperature at which virus was grown. Revertants at position 472 of type 3 OPV accumulated faster in vaccines derived from Sabin Original (SO) substrain than from RNA-plaque purified (RSO) substrain. The rate of accumulation of 472-C revertants differed among cell lines and was higher in overgrown cell cultures suggesting that host factors are involved in the selection of mutants. We also found that accumulation of mutants occurred in vitro at position 480 in type 1 and position 481 in type 2 OPV, making the selection for revertants in domain F of the 5'-noncoding region a general phenomenon for all three Sabin strains. Assessment of the abundance of these mutants may be used for evaluation of the quality of OPV lots.