Two approaches were used to demonstrate proteolysis of reovirus in the intestine of the neonatal mouse. The first approach utilized peroral inoculation of radiolabeled virus into neonatal mice; the intestinal washings were harvested at 0 to 30 min postinoculation. The virus recovered from the intestinal washings was electrophoresed in polyacrylamide to determine whether proteolytic digestion of viral proteins had occurred. Complete loss of sigma 3 and generation of the mu 1c cleavage product delta demonstrated that digestion occurred within 10 to 30 min after the inoculation, resulting in the rapid generation of intermediate subviral particles (ISVPs). The products formed resembled those seen when the virus is digested in vitro with chymotrypsin. The second approach took advantage of the fact that ISVPs grow in cells treated with NH4Cl, whereas intact virus does not grow under these conditions (L. J. Sturzenbecker, M. Nibert, D. Furlong, and B. N. Fields, J. Virol. 612351-2361, 1987). Thus, assaying virus for its ability to grow in NH4Cl-treated cells represents a means of ascertaining whether the samples contain ISVPs. Using this approach, we demonstrated that up to 8 h postinoculation ISVPs predominate in the intestinal tissue and in the intestinal lumen. Between 8 and 15 h postinoculation, there is a loss in the proportion of ISVPs in the tissue so that by 15 h postinoculation ISVPs are no longer detectable in intestinal tissue washed of lumen contents and virus. In contrast, the lumen of the intestine contains some ISVPs at all times postinoculation. Thus, after peroral inoculation, the mammalian reoviruses are converted to proteolytically cleaved virus, suggesting that proteolysis plays an important role in initiation of infection in the gastrointestinal tract.