The genetic and physical structures of commonly used F-prime factors carrying the galactose region of the Escherichia coli chromosome were analyzed. Deletions in the chromosomal DNA sequences in the F-prime factors were found to be frequent events. A genetic method was developed to reconstruct the original F-prime factors from deletion variants. Heteroduplex analysis of the reconstructed F-prime factors confirmed the derivation of the F-prime factors F100 and F152, from the same Hfr, and finally determined the normal E. coli chromosomal sequence in the region between fep and uvrB, containing about 5 min in genetic units and about 246.5 in kilobase units (kb). This sequence could be connected with the DNA sequences of the lac-purE region, which had been physically determined previously. Together they constituted a total of 528.6 kb. From these combined sequences, the distance from lacPO to galK was calculated to be 412.9 kb, which corresponds to 8.8 min in genetic units.