A promoterless gene for the Shiga toxin 1 B subunit (stxB1) has been placed under transcriptional control of the Vibrio cholerae heat shock gene htpG. A chromosomal enterohemorrhagic Escherichia coli fragment containing eaeA and 400 bp of upstream DNA was added to the construct, downstream of stxB1; no transcription terminators were located between the two genes. The plasmid construct was confirmed by DNA sequencing; in vitro transcription-translation studies demonstrated expression of EaeA from the plasmid. The htpGp-->stxB1, eaeA construct was inserted into lacZ on the chromosome of Peru2, an El Tor V. cholerae strain with both attRS1 sequences and the entire cholera toxin genetic element deleted, and into lacZ in JRB10, a Peru2 derivative that has a second copy of htpGp-->stxB1 also inserted in the V. cholerae virulence gene irgA. Two plasmid constructs, one containing stxB1 under the control of the tac promoter and another containing htpGp-->stxB1,eaeA, were transformed into Peru2. Expression of StxB1 by these constructs was quantified by enzyme-linked immunosorbent assay and was highest in the plasmid construct with stxB1 under the control of the tac promoter. Localization of EaeA to the outer membrane of the vector strains was demonstrated both by Western blotting and by immunofluorescence with an anti-EaeA antibody. A rabbit model for colonization by V. cholerae was used to compare the immune responses to the two heterologous antigens, StxB1 and EaeA, expressed by these strains. Rabbits immunized with Peru2 transformed with a plasmid carrying tac-->stxB1 developed neutralizing serum anti-StxB1 immunoglobulin G antibody responses. One of two rabbits immunized with a strain carrying a chromosomal copy of eaeA developed a marked immune response against EaeA. The plasmid construct containing htpGp-->stxB1,eaeA was unstable, producing low levels of StxB1 in vitro and not evoking anti-EaeA antibody responses in vivo following oral immunization. Chromosomal insertion of eaeA may be preferred for future expression of this antigen in V. cholerae vaccine constructs.