T lymphocytes play an important role in host responses to microbial antigens, and therefore, in order to facilitate studies to determine the mechanisms involved in the regulation of these responses, a method for the generation of T cell clones with specificity to microbial whole cell (WC) antigens has been developed. T cells were purified from the spleen of rats immunized with WC antigen of either the gram-positive bacterium Streptococcus mutans or the gram-negative bacterium Bacteroides gingivalis and cultured with irradiated Sephadex G-10-passed spleen (feeder) cells and the homologous WC antigen for the generation of T cell clones. Clonal growth of the T cells was maintained for up to 6 months by co-culturing similar numbers of T cells, feeder cells and WC antigen; however, the addition of exogenous interleukin-2 (IL-2) was not required. Phenotypic characterization of the cloned T cells showed that they were CD4+ T helper (Th) cells which did not express the leukocyte-common antigen, but did express receptors for IL-2. These T cells required the presence of homologous WC antigen for growth and their antigen specificity was further confirmed by the ability of the T cells to proliferate in response to the homologous WC, but not to heterologous microbial WC, antigen. These T cells were further characterized for their ability to produce cytokines, specifically IL-2 and interferon-γ, and to provide help for B cell responses to microbial WC antigen. This study describes a reproducible method for the generation of rat Th cell clones with specificity to microbial WC antigens which will be valuable for defining the mechanisms involved in T cell regulation of responses to either gram-positive or gram-negative bacteria. © 1991.