The genes controlling synthesis of Shigella flexneri group- and type-specific antigens were transferred to Escherichia coli K-12 recipients by conjugation with an S. flexneri Hfr. After mating E. coli with an Hfr strain of S. flexneri 2a and selecting for his(+) recombinants, a high proportion of the E. coli hybrids agglutinated in S. flexneri grouping serum. None of these hybrids expressed S. flexneri type-specific antigen II. When an E. coli his(+) hybrid possessing the S. flexneri group antigen was remated with the same Hfr with selection for pro(+) hybrids, a high proportion now expressed the type-specific antigen as well as the previously inherited group antigen. If such crosses were performed in reverse order (i.e., pro(+) followed by his(+) selection), a different pattern of serological behavior was observed. None of the pro(+) hybrids showed the type-specific antigen. Subsequent mating for his(+) resulted in hybrids with both the group- and type-specific antigens. These results show that genes controlling the synthesis of S. flexneri group antigen (linked to the his locus) and type-specific antigen (linked to the pro locus) are widely separated on the chromosome. Expression of the type-specific antigen II depends on the presence of the group antigen.