Normal human beings have circulating T lymphocytes that proliferate in response to Escherichia coli and Pseudomonas aeruginosa. We performed the present study to characterize the nature of the responding T cells and to determine whether distinct or shared conventional antigens, superantigens or polylonal activators account for T cell proliferation. Long term antigen‐specific T cell lines were generated by repeated stimulation of PBMC from four donors with soluble antigen preparations of E. coli or P. aeruginosa. This resulted in the emergence of distinct T cell populations, which responded to strains of either E. coli or P. aeruginosa, but not to both. Trypsin treatment of the bacterial preparations largely eliminated their ability to stimulate the T cells. The T cell lines were predominantly CD4⁺ and their proliferation to bacterial antigens was optimal using autologous APC. E. coli T cell lines proliferated not only in response to the E. coli strain with which they were initially selected, but also to four different strains of E. coli, as well as to several related Gram‐negative species. P. aeruginosa selected T cells exhibited proliferative responses to six different P. aeruginosa strains, but not to the other Gram‐negative species. The finding that repeated stimulation of PBMC with E. coli or P. aeruginosa leads to CD4⁺ T cells highly reactive with conventional protein antigens specific either for E. coli or P. aeruginosa indicates that these bacteria possess separate dominant protein antigens that drive the proliferation of peripheral blood T cells.