The unique murine lymphocyte differentiation antigen, Lp-3, with a mol. wt of approximately 125 kd, was found using a rat monoclonal antibody. The Lp-3 antigen was distributed on a wide variety of myeloid, T cell, and B cell lineages in mice. However, the expression was only found in B cells at certain stages of differentiation. The pre-B and virgin B cells in the bone marrow from 2-month-old BALB/c mice were weakly positive for Lp-3, while the resting B cells in the spleen and lymph node were Lp-3 negative. In contrast, the majority of B cells in the peritoneal cavity, mostly Ly-1 (CD5) B cells, had a brighter fluorescence for Lp-3 than did bone marrow B cells. The Lp-3 antigen could be induced in a high density in approximately one-half of lipopolysaccharide-stimulated, large, blastic spleen B cells. Cell cycle analysis showed that Lp-3 is an early B cell activation antigen which is first expressed at the G1A phase of the cell cycle. Therefore this novel B cell differentiation antigen will be useful for differentiating pre-B and virgin B cells in the bone marrow, resting B cells, and a population of activated B cells in the periphery. In contrast to findings in BALB/c mice, there was an elevated population of B cells with a bright Lp-3 expression in the spleen of autoimmune-prone NZB x NZW F1 mice.