The initial rates of induced synthesis of tryptophanase, beta-galactosidase, and d-serine deaminase were measured in relation to the chromosome replication cycle of Escherichia coli B/r. Exponentially growing cultures were exposed briefly to (14)C-thymidine or the appropriate inducers (or both), and the amount of label or enzyme (or both) in cells of different ages was found by measuring these quantities in their progeny. The rates of induced synthesis of the three enzymes increased abruptly at about 4, 20, and 34 min, respectively, after the start of a round of replication lasting 40 min. By matching this sequence to the ind, lac, and Dsd loci on the genetic map of E. coli K-12, it was estimated that replication began at about 8 o'clock (60 min) and proceeded clockwise. In rapidly growing cells, the sequence during the division cycle was consistent with the concept that rounds of replication overlapped.