Several conditional-lethal mutations that do not permit the replication of F-factors of Escherichia coli K-12 are located at a site called seg. This gene is located on the E. coli chromosome between serB and thr. It is unrelated to other known genes involved in DNA replication. Strains carrying seg mutations were unable to replicate F′-lac+, several F′-gal+s, F′-his+ and bacteriophage λ at 42°. However, neither phage T4, ColE1, nor any of the R factors tested were prevented from replicating at 42°C. When the kinetics of the loss of F-primes is studied in seg strains, it is found that the rate of curing depends on the size of the plasmid, larger F factors curing faster than smaller ones, and that Hfrs are formed at high frequencies. The Hfrs showed both F-genote enlargement and normal transfer of chromosomal markers. The F-genotes are unstable and segregate chromosomal markers at high frequencies. Some orthodox Hfrs were examined, and two that were known to revert to the F+ condition relatively frequently were found to generate enlarged F-genotes on mating, whereas two strains that were very stable with respect to reversion to the F+ state did not show F-genote formation. F-genote formation from seg Hfr strains is dependent of a functional recA gene, as F-genote formation was not seen with a seg-2, recA-1 Hfr. This is in contrast to F-genote enlargement shown by both orthodox Hfrs and an Hfr strain constructed by integration of a temperature-sensitive F′-gal+, whose F-genote enlargement is Rec-independent. Thus there may be more than one mechanism for the formation of enlarged F-genotes.