Murine leukemia virus (MuLV) genome encodes a protease (Y. Yoshinaka, I. Katoh, T.D. Copeland, and S. Oroszlan (1985), Proc. Natl. Acad. Sci. USA 82, 1618-1622), which has been shown to cause maturation, specified as morphological conversion from "immature" to "mature" form of virus cores. To examine whether "immature" particles have infectivity or not, we constructed mutant DNAs with deletions in the protease region. The NIH/3T3 cells transfected with mutant DNAs produced "immature" particles, having immature morphology and containing Pr65gag, a polyprotein precursor of core proteins. The specific infectivity of the extracellularly released and purified particles was shown to be greatly reduced based on reverse transcriptase activity and protein content as compared with the "mature" particles obtained from wild-type DNA-transfected cells. The mutant genomes encoded functionally normal surface glycoprotein, gp70. These results strongly suggest that maturation of MuLV from "immature" to "mature" form of virus particles is indispensable to virus infectivity. The importance of processing of gag and pol, as well as transmembrane protein precursors by the viral protease is discussed.