The three serotypes of reovirus differ markedly in their response to a variety of chemical inactivating agents. We used intertypic recombinants containing various combinations of genes derived from the parental serotypes to study the basis of these differences. In addition to recombinants derived from types 1 and 3, and 2 and 3, we were able to isolate recombinants derived from types 1 and 2, suggesting that these two serotypes also undergo unrestricted reassortment. The intertypic recombinants behaved like one parent or the other in the presence of the inactivating agents and allowed us to determine the genes responsible for each difference. Recombinants derived from crosses between wild-type parental serotypes produced straightforward results, while recombinants derived from mutagenized, temperature-sensitive parents often did not. Sensitivity to 2.5 M-guanidine-HCl and pH 11 was determined by the S1 gene, sensitivity to 55 degrees C and 1% SDS was determined by the S4 gene, and sensitivity to 33% ethanol and to 1% phenol was determined by the M2 gene. Thus, relatively nonspecific chemical agents appear to have their predominant effect on specific proteins of the reovirus virion.