Two heat-sensitive R.BamHI mutants, T1571 and P173L, and one cold-sensitive R.BamHI mutant, T1141, were isolated after chemical mutagenesis of the bamhIR gene that codes for the restriction endonuclease BamHI (R.BamHI). The thermosensitivity of T1141, T1571 and P173L is revealed by the 10 2−10 3 lower plating efficiency at the non-permissive temperature of strains bearing these alleles. The conditional-lethal phenotype can be rescued by introduction of the cognate bamhIM gene into the same cell. The mutant enzymes induce the SOS response in vivo and display reduced phage restriction activity. The P173L protein, when expressed at 30°C and purified, shows reduced thermostability at 65°C. T1571 and P173L mutants yield different intermediates during partial trypsin digestion. The conditional-lethal BamHI mutants could be used to deliver in vivo DNA cleavage and for further isolation of relaxed-specificity mutants.