Stable self-reactive T-cell lines were established from the draining lymph nodes of Buffalo rats primed with rat thyroglobulin (Tg) in complete Freund's adjuvant. Following two rounds of in vitro selection with Tg or with purified protein derivative (PPD) of Mycobacterium tuberculosis, Tg- or PPD-specific lines were selected. Self-reactivity, manifested by a potent proliferative response of these lines to syngeneic stimulators in the absence of the selecting antigen (Tg or PPD), emerged by 3-4 weeks after the establishment of the lines. This proliferation could not be attributed to reactivity against heterologous serum components. Despite this syngeneic mixed-leukocyte reaction (SMLR) which was readily detectable, specific stimulation by Tg or PPD over the level of the SMLR could still be demonstrated. These lines also responded to concanavalin A but not to allogeneic stimulator cells. More than 95% of T cells of these lines displayed the rat T-helper-cell-specific (W3/25) marker and they secreted a defined lymphokine, T-cell growth factor (TCGF), upon stimulation with syngeneic stimulator cells, independent of the presence of soluble antigen. T-Cell lines such as the ones described here will be useful in elucidating the biological significance of the AMLR/SMLR and its relation to nominal antigen reactivity.