We have determined the origin and cell surface phenotype of B cells producing antibody in response to immunization with the non-self TI-2 antigen polyvinyl pyrrolidinone (PVP). We report that the responding cells are derived from precursors in adult bone marrow and display the phenotype characteristic of B-1 cells. By use of allotype marked chimeric mice, constructed by reconstituting lethally irradiated recipients with adult bone marrow and peritoneal B-1 lymphocytes of recognizably different Ig allotypes, immunized with 1 microgram PVP, we found that although a substantial part of the total IgM produced in these chimeras bore the allotype of the transferred peritoneal B-1 cells, essentially all of the anti-PVP IgM expressed the allotype of the adult bone marrow. Fifteen of 16 hybridomas derived from a normal PVP-immune adult mouse bore N nucleotides at the V-D and D-J junctions of their heavy chain CDR3 regions, indicating their origin from precursors in the adult bone marrow. By use of ELISA spot analysis, we found the cells responding to PVP to be localized in the spleens of normal immunized mice. We then used multiparameter flow cytometric sorting to determine the cell surface phenotype of these cells. We found that the cells producing anti-PVP were greatly enriched in a small subpopulation with the phenotypic characteristics of B-1 cells; they were B220intermediate, CD5low, IgMhigh, IgDlow, CD43+ and CD23-. This subpopulation was also enriched for all cells producing IgM, regardless of specificity (the so-called 'spontaneous' antibody). We conclude that the B-1 phenotype is more likely a marker for a state of differentiation than for a discrete lineage of B cells.