Localized P1 mutagenesis has been used to isolate conditionally lethal mutations in the four-minute region of the Escherichia coli genome. One such mutation, ts25, has been mapped at about 3.7 minutes between the popC and dapD genes. This mutation leads to thermosensitivity of growth and impaired in vivo assembly of 30 S ribosomal subunits at 42 °C. The strain carrying the mutation has an altered S2 ribosomal protein as judged by (1) its inability to maintain stable complex with the ribosome under mild washing conditions and (2) its altered electrophoretic mobility. Spontaneous reversion to temperature independence can restore both the normal assembly in vivo of 30 S ribosomal subunits at 42 °C and the normal electrophoretic behaviour of the S2 ribosomal protein in vitro. We conclude therefore that the ts25 mutation affects the structural gene for ribosomal protein S2 (rpsB).