Maturation of picornaviruses involves assembly of a "provirion," which undergoes an autocatalytic cleavage of VP0 to VP2 plus VP4. RNA transcripts from a cDNA clone of human rhinovirus 14 mutated at asparagine 68, one of the residues in the maturation cleavage site, generated normal yields of 150S particles which were noninfectious in the plaque assay because they were unable to initiate a second cycle of infection. These cleavage-defective provirions were otherwise indistinguishable from mature virions in sedimentation coefficient, binding affinity to monoclonal antibodies against neutralization sites IA, II, and III, attachment to HeLa cell receptors, and rate of cell-mediated conformational changes to form 125S A-particles and 80S empty capsids. These results suggest that maturation cleavage is required for the function of a previously undescribed intermediate which transfers packaged RNA across the membrane and into the cytosol. For this hypothetical intermediate, we propose the name infectosome. Since the native virus has a particle/PFU ratio of about 800, such an intermediate will be difficult to find. Mutations at serine 10 in VP2 reduced maturation cleavage to a rate sufficiently slow to show that the infectivity of virus particles increased with the degree of cleavage of VP0 to VP4 and VP2. This article describes the first characterization of a pure form of a picornaviral provirion, and hence the first direct evidence that provirions of picornaviruses lack infectivity.