Targeted mutation of the gene for the Oct-2 transcription factor in mice caused neonatal lethality and abrogated mitogen-induced proliferation and differentiation of mature B lymphocytes in vitro. Here we show that Oct-2 is required for normal humoral responses upon immunization with T cell-dependent as well as T-independent Ags. oct-2-null T cell behavior was normal, implying a B cell-restricted lesion. oct-2-/- B cells displayed aberrant behavior during activation in vitro: both acquisition of markers of cellular activation and cell survival were diminished. Production of early B lineage cells in the bone marrow was normal, yet mature B cells were under-represented in blood and lymphoid organs. Furthermore, peritoneal B-1 lymphocytes were not detected in animals with a reconstituted oct-2-/- lymphoid system. We conclude that Oct-2 is required for B-1 cell maintenance and for normal Ag-driven maturation of conventional B cells in vivo.