Shigella and enterotoxigenic Escherichia coli continue to be significant causes of diarrheal disease in infants and young children in developing countries as well as prevalent agents of traveler's diarrhea. A vaccine which provides protection against disease caused by both pathogens would serve common at-risk populations. Such a vaccine would require inclusion of multiple Shigella strains as well as multiple ETEC antigens. The use of attenuated strains of Shigella as live vectors for the expression of ETEC antigens is one strategy for the development of such a multivalent vaccine. Live attenuated strains of S. flexneri 2a, S. sonnei and S. dysenteriae 1 containing deletions in guaBA biosynthetic pathway genes as well as in genes encoding enterotoxins, were constructed. Each strain was subsequently used as a live vector for the expression of one or two critical ETEC antigens. The resulting three Shigella derivative strains were tested for immunogenicty and protective capacity alone or as mixtures in the guinea pig model. S. flexneri strain CVD 1208(pCFA/I-CS3), S. sonnei strain CVD 1233(pCS4-LThK63) and S. dysenteriae 1 strain CVD 1252(pCS2) were able to elicit serum and mucosal antibody responses against the live vector as well as the guest ETEC antigens. Vaccination with combinations of two or three of these strains was able to elicit specific immune responses against each live vector as well as each ETEC antigen represented in the mixture. These studies demonstrate the potential of the use of mixtures of live Shigella derivatives expressing ETEC antigens to serve as an immunogenic multivalent vaccine.