We have produced mice that lack major histocompatibility complex class II antigens, permitting us to evaluate the role of these molecules in diverse aspects of T and B cell differentiation. The mutant mice show near-complete elimination of CD4+ T lymphocytes from the spleen and lymph nodes; the few remaining CD4-positive cells are preferentially localized to B cell follicles. Surprisingly, substantial numbers of CD4 single-positive cells reside in the thymus; however, these are not mature thymocytes as we currently recognize them. B lymphocytes occur in normal numbers and are capable of terminal differentiation to plasma cells. Nevertheless, several aberrations in the B cell compartment are demonstrable: a lack of germinal centers, fewer IgM+IgD+ cells in certain individuals, reduced production of serum IgG1, and complete inability to respond to T-dependent antigens. In short, the class II-negative mice have confirmed some old ideas about lymphocyte differentiation, but have provided some surprises.