We report the isolation of E. coli mutant capable of supporting replication of bacteriophage Qbeta at 33 degrees, but not at 40 degrees. Coliphages f2, R23, fd, and yamma formed plaques on mutant cells at both temperatures. Temperature-shift experiments showed that bacteriophage Q beta replication was blocked in the mutant within the first 20-30 min of infection. The defect did not prevent translation of the Qbeta polymerase gene or assembly of catalytically active Qbeta replicase molecules. In fact, mutant cells infected at 40 degrees hyperinduced replicase active both in vivo and in vitro. However, zone sedimentation of the in vivo RNA product showed it to consist of partially double-stranded material sedimenting at 9 S, with little or no viral 32S RNA. The 9S RNA was also found, along with a predominant peak of 32S RNA in parental cells infected at 40 degrees, but not in cells infected at 33 degrees. It thus appears that the temperature-sensitive component is required for viral RNA replication, but not for other RNA synthesis catalyzed by the replicase. Uninfected mutant cells grew normally at 40 degrees in nutrient broth, but not in glucose- or glycerol-minimal media. Revertants selected for their abillity to grow in minimal medium at 40 degrees also supported bacteriophage Qbeta replication at 40 degrees.