The majority of clonotypic CD4+ T cells in the intestinal lamina propria of DO11.10 TCR transgenic mice have an activated/memory phenotype and produce effector cytokines despite the absence of prior exposure to ovalbumin (OVA), the transgene-specific antigen. A small number of splenic T cells have a similar phenotype. Clonotypic T cells from Peyer's patch are intermediate in both phenotype and effector cytokine production. Flow cytometric analysis of cells isolated from thymectomized, OVA-naive DO11.10 mice treated with continuous administration of BrdU indicated that a significant fraction of clonotype-positive T cells in the lamina propria and Peyer's patch were in the cell cycle, with significantly fewer cycling cells in the spleen. Most of the cycling cells from each anatomic site expressed low levels of CD45RB. Effector cytokine expression was enriched in the CD45RB(low) populations. These memory/effector cell populations were eliminated in DO11.10/SCID and DO11.10/RAG-2(-/-) mice, suggesting that recognition of non-OVA antigens through a second, non-clonotypic TCR was driving differentiation of memory/effector cells in naive BALB/c DO11.10 mice. Clonotypic CD4+ T cells isolated from DO11.10, but not from DO11.10/SCID or DO11.10/RAG-2(-/-) mice, were stimulated to enter the cell cycle by antigen-presenting cells pulsed with an intestinal bacterial antigen extract. These data provide direct evidence that enteric bacterial antigens can activate transgenic T cells through a second, non-clonotypic TCR, and support the notion that the development and turnover of memory/effector cells in vivo is driven by the intestinal flora.