The infectivity of rotavirus particles is dependent on proteolytic cleavage of the outer capsid protein, VP4, at a specific site. This cleavage event yields two fragments, identified as VP5* and VP8*. It has been hypothesized that the particle is more stable, but non-infectious, when VP4 is in the uncleaved state. Uncleaved VP4 and the resultant increased stability might be advantageous for the virus to resist environmental degradation until it infects a susceptible host. When VP4 is cleaved in the lumen of the host's gastrointestinal tract, the virus particle would become less stable but more infectious. To test this hypothesis, a series of experiments was undertaken to analyse the cleavage state of VP4 on virus shed by an infected host into the environment. Immunoblots of intestinal wash solutions derived from infant and adult BALB/c mice infected with a virulent cell culture-adapted variant of the EDIM virus (EW) or wild-type murine rotavirus EDIM-Cambridge were analysed. Virtually all of the VP4 in these samples was in the cleaved form. Moreover, cell culture titration of trypsin-treated and untreated intestinal contents from pups infected with EW indicated that excreted virus is fully activated prior to trypsin addition. It was also observed that trypsin-activated virus has no disadvantage in initiating infection in naive animals over virions containing an intact VP4. These studies indicate that VP4 is cleaved upon release from the intestinal cell and that virus shed into the environment does not have an intact VP4.