The type I restriction and modification genes of Escherichia coli can be transferred to other non-modified strains by conjugation without killing the recipient, implying that the restriction function must be regulated. In this study, two isogenic F plasmids (r _K ⁺ and (r _K ^– served as donors in quantitative conjugation experiments with various restriction-deficient strains of E. coli and Salmonella typhimurium. Conjugation studies with hsd::lacZ operon fusions in F plasmids indicate that both of the hsd _K promoters, p _res, and p _mod, express simultaneously following conjugative transfer. Thus these genes do not appear to be regulated at the transcriptional level. A spontaneous mutant of E. coli C was discovered that is presumably killed upon conjugative transfer of the hsd _K genes (defined as a Crc^– phenotype). The gene that is defective in the mutant was tentatively designated hsdC (control). Hfr gene replacement studies led to the localization of the putative hsdC gene between 6 and 16 min on the E. coli genetic map.