The use of attenuated strains of Salmonella as vaccine vectors frequently involves the introduction of heterologous antigens on recombinant plasmids. To overcome the problem of spontaneous loss of the plasmid, we have integrated the gene that codes for a potential immunogen into the chromosome of a galE mutant of S. typhimurium. Comparative in vitro and in vivo studies were conducted between the strain carrying the gene chromosomally integrated, and an isogenic strain carrying the same gene on a multicopy plasmid. Levels of expression of the foreign antigen were significantly lower when the antigen was expressed from a single copy of the gene on the chromosome, than when it was expressed from the multicopy plasmid. The in vivo stability of antigen expression was determined on organisms recovered from spleen, liver and Peyer's patches of orally inoculated mice. By 24 hours post inoculation, the majority of tissue isolates from the plasmid containing strain had lost the plasmid and the ability to synthesize the antigen in vivo. By contrast, 100% of the cointegrate isolates expressed the antigen in vivo throughout the 21 days of the experiment. Significantly, humoral and mucosal antibody levels against the antigen were greater when the antigen was expressed from the plasmid stabilized by the presence of the antibiotic than when the antigen was expressed from the chromosome. These observations indicate that an important event for the development of an immune response against a foreign antigen delivered by these vectors is the initial amount of antigen that primes the GALT