A small number of B cells are found in the thymus of normal mice. A population of B lymphocytes could be enriched to greater than 90% purity by isolating a low-density fraction on Percoll density gradients and then depleting T cells with a mixture of anti-Thy-1, CD4, and CD8 mAbs and complement. Enrichment was monitored by surface Ig staining and by functional studies (responsiveness to LPS, and to anti-mu plus IL-4). When the phenotype of these B cells was studied by flow cytometry, 60-80% had the phenotype Ly-1+ (CD5), Ia+, B220low (CD45R), and Mac-1+ (CD 11b). In contrast, splenic B cells lacked CD5 and CD11b and expressed higher levels of B220 and Ia antigens. These results indicate that most thymic B cells have the phenotype of the Ly-1 B cell subset, which was identified previously as a trace subpopulation in some peripheral tissues and is thought to play a role in autoantibody formation.