Cell entry by reoviruses requires a large, transcriptionally active subvirion particle to gain access to the cytoplasm. The features of this particle have been the subject of debate, but three primary candidates-the infectious subvirion particle (ISVP), ISVP*, and core particle forms-that differ in whether putative membrane penetration protein micro 1 and adhesin sigma1 remain particle bound have been identified. Experiments with antibody reagents in this study yielded new information about the steps in particle disassembly during cell entry. Monoclonal antibodies specific for the delta region of micro 1 provided evidence for a conformational change in micro 1 and for release of the delta proteolytic fragment from entering particles. Antiserum raised against cores provided evidence for entry-related changes in particle structure and identified entering particles that largely lack the delta fragment inside cells. Antibodies specific for sigma1 showed that it is also largely shed from entering particles. Limited coimmunostaining with markers for late endosomes and lysosomes indicated the particles lacking delta and sigma1 did not localize to those subcellular compartments, and other observations suggested that both the particles and free delta were released into the cytoplasm. Essentially equivalent findings were obtained with native ISVPs and highly infectious recoated particles containing wild-type proteins. Poorly infectious recoated particles containing a hyperstable mutant form of micro 1, however, showed no evidence for the in vitro and intracellular changes in particle structure normally detected by antibodies, and these particles instead accumulated in late endosomes or lysosomes. Recoated particles with hyperstable micro 1 were also ineffective at mediating erythrocyte lysis in vitro and promoting alpha-sarcin coentry and intoxication of cells in cultures. Based on these and other findings, we propose that ISVP* is a transient intermediate in cell entry which mediates membrane penetration and is then further uncoated in the cytoplasm to yield particles, resembling cores, that largely lack the delta fragment of micro 1.