Abstract In a dna C ts mutant of E. coli , the reinitiation of DNA synthesis, which occurred by the shift of the culture from a restrictive temperature to a permissive temperature, was markedly prevented by habakacin, dibekacin, kanamycin, and gentamicin. On the contrary, chloramphenicol did not inhibit the reinitiation synthesis for 30 min. In a parallel experiment, leucine uptake into protein was profoundly blocked by chloramphenicol, but only slightly by habekacin. Habekacin did not significantly affect DNA elongation of the cells at a restrictive temperature. We propose that inhibition of initiation of replication by aminoglycoside antibiotics is related to their lethality.