A mutant of human immunodeficiency virus type 1 (HIV‐1), which is deficient for Gag precursor cleavage and non‐infectious, was characterized with respect to its defective step in the viral replication phase. Upon transfection, the mutant produced a normal level of progeny virions as monitored by electron microscopy and RNA hybridization. Single‐round replication assay demonstrated, in contrast, that the mutant was defective at the early phase of the replication cycle. Furthermore, no viral DNA was detected in the cells infected with the mutant. Taken together, it is concluded that maturation of Gag precursor protein of HIV‐1 is required for an early event(s) before or during a coupled process of uncoating/reverse transcription.