Several conditional-lethal mutantions 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 ser B 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 gamma at 42 degrees. However, neither phage T4, ColE1, nor any of the R factors tested were prevented from replicating at 42 degrees 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 the F+ state did not show F-genote formation. F-genote formation from seg Hfr stains is dependent on 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.