Eighteen mutants that are temperature-sensitive for vegetative replication (rep) were isolated from two F′-gal+ plasmids (F8 and F8-4) after N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis. Some of the mutants also have reduced transfer ability at both permissive and nonpermissive temperatures. Plasmid-plasmid P1 transduction has revealed that in some instances, the altered transfer ability is located in the transfer operon and is distinct from the rep mutation. However, in other cases, the replication and transfer defects have not been separated by P1 transduction. The implications of these results for the relationship between vegetative DNA replication and DNA replication during conjugation are discussed. In vivo recombinational results suggested that the temperature-sensitive mutations were not located in the same regions of the two F′-plasmids. We confirmed that no inversion, secondary deletion, or translocation of DNA had occurred in either F8 or F8-4, and suggest that the apparent difference is due to a recombination anomaly.