Due to critical amino acid changes in the 72-89 sequence, the determinant of human (Hu) basic protein (BP) that induces experimental autoimmune encephalomyelitis (EAE) in Lewis rats most likely differs from rat and guinea pig BP. To discern encephalitogenic sequence(s), the immunodominant epitopes recognized by Hu-BP-specific T cell lines were identified using synthetic peptides that corresponded to the Hu-BP sequence. The Hu-BP-reactive T cell line contained two distinct specificities, one directed at the 87-99 (Hu) sequence restricted by I-E, and the second directed at the 55-74 (Hu) sequence restricted by I-A. T cells specific for the 87-99 determinant recognized both Hu- and Rt-BP, were highly encephalitogenic, and accounted for the experimental autoimmune encephalomyelitis-inducing activity of the Hu-BP line. T cells directed at the S55-74 (Hu) sequence did not recognize Rt-BP and were not encephalitogenic. The same TCR V genes (homologous to the mouse V alpha 2 and V beta 8 families) that we showed previously were utilized preferentially in response to the I-A restricted 72-89 encephalitogenic sequence were also present in T cell lines specific for both the S55-74 and S87-99 epitopes. These data indicate that encephalitogenic activity of BP in Lewis rats is related to discrete T cell epitopes that are present on or cross-react with rat-BP. Furthermore it would appear that genes in the TCR V alpha 2 and V beta 8 families are widely used in response to different BP epitopes restricted by either I-A or I-E molecules.