During the past several years, much interest has been directed towards delineating and characterizing different subsets of T helper (Th) cells in order to understand their roles in immune processes. In this study, we report the generation of antigen-specific rat Th cell clones and their characterization in terms of phenotype, function, and lymphokine production. The clones were derived by culturing purified splenic T cells from rats immunized with the pathogen Bacteroides gingivalis with equivalent numbers of irradiated spleen cells from nonimmune rats and B. gingivalis whole-cell antigen. The clones required antigen stimulation but not exogenously added interleukin-2 for growth and were maintained in culture for approximately 6 months. The cloned T cells proliferated in response to the mitogen concanavalin A and to B. gingivalis whole-cell antigen but not to other microbial antigens. Phenotypic characterization of the cloned T cells for cell surface markers demonstrated that these cells were OX19+ W3/25+ OX8- OX22- and therefore probably represented a mature subpopulation of CD4+ Th cells. These cloned T cells were positive for interleukin-2 receptor expression. Culture supernatants from the Th cell clones which were collected at various times after antigen stimulation exhibited low interleukin-2 activity and high gamma interferon activity. This in vitro study provides evidence of a rat Th cell subset that could represent an important population in regulating immune responses to microbial antigens.