More temperature-sensitive mutants affecting the replication of the F′-gal+ episome of Escherichia coli K12 have been isolated. Eight of the mutations were located on F itself and three were located on the chromosome. The temperature sensitive F′-gal+'s have been integrated into the chromosome to produce Hfr strains. These Hfr strains have transfer origins similar to Hfr Cavalli, and all show aberrant excision and transfer of elongated segments of the chromosome including the integrated F-gal to generate long merodiploids. The chromosomal mutations that govern the replication of F have been termed seg (for segregation). Wild-type F-gal+ can be integrated into seg cells at 42° C to give Hfrs, in a process analogous to integrative suppression in the formation of Hfrs from cells carrying mutations that are temperature-sensitive for chromosomal DNA replication (dnaA). A curious feature of an Hfr derived from a seg strain is that it also shows F-genote enlargement as well as normal transfer of chromosomal genetic marker. Preliminary transductional mapping data show that the mutation seg-2 is linked to the threonine locus (minute 0).