Investigations were carried out to determine whether both DNA strands involved in Escherichia coli chromosomal DNA replication are replicated with similar accuracy. Experiments consisted of measuring the forward mutation rate from tonB(+) to tonB(-) in pairs of polA deficient strains in which the chromosomal target gene tonB was oriented in the two possible directions relative to the origin of replication, oriC. Within these pairs, the tonB sequence would be subjected to leading strand replication in one orientation and to lagging strand replication in the other. The most common tonB mutations in the polA1 strain were deletions followed by frameshifts. Among the deletions, a strong hotspot site with a 13-base deletion in the polA1 strains accounted for 18 of the 33 deletions in the one orientation, and 31 of the 58 deletions in the other. The results suggested that the two strands were replicated with equal or similar accuracy for deletion formation.