In an effort to elucidate the role of intrathymic Ia-bearing antigen-presenting cells (APC) on the development of the class II-restricted T cell repertoire, we examined the effect of neonatal anti-I-A treatment on both intrathymic and splenic APC function; on the generation of Lyt-2-,L3T4+, Lyt-2+,L3T4-, and Lyt-2+,L3T4+ T cells; and on the development of class I- and class II-specific T cell functions. Both the thymus and the spleen are completely devoid of Lyt-2-,L3T4+ T cells in young mice treated from birth with anti-I-A, and also lack functions associated with this subset, i.e., alloantigen-specific interleukin 2 production (present report), allo-class II-specific and self-class II-restricted T cell proliferative responses, and helper cell function for the generation of cytotoxic T lymphocyte responses (18). Development of the Lyt-2+,L3T4- subset proceeds undisturbed in these mice, in accord with the previously reported normal levels of cytotoxic T lymphocyte precursors (18). The thymus contains normal numbers of the immature cortical Lyt-2+,L3T4+ cells, indicating that acquisition of the L3T4 marker, in and of itself, is not influenced by anti-I-A treatment. This striking absence of the lineage of T cells responsible for class II-specific T cell functions is correlated with absence of thymic APC function for class II-restricted T cell clones. When anti-I-A-treated mice are allowed to recover from the antibody treatment, splenic and thymic APC function return to normal in 2-3 wk, and thymic Lyt-2-,L3T4+ T cell numbers and functions reappear before such cells are detectable in the spleen. Collectively, these findings suggest that development of the Lyt-2-,L3T4+ lineage of class II-specific T cells is entirely dependent on functional I-A-bearing APC cells in the thymus. In addition, the presence of normal levels of Lyt-2+,L3T4-T cells argues that generation of the two major subsets of T cells (i.e., Lyt-2+,L3T4- and Lyt-2-,L3T4+) occurs through separate events, involving unique sites of interactions between precursor T cells and nonlymphoid major histocompatibility complex-bearing thymus cells.