A monoclonal antibody (mAb), Hy30, was generated against bursal cells. Under reducing conditions, it immunoprecipitates a cell surface antigen with an apparent molecular weight of 66 kD that is distinct from immunoglobulin and MHC class II. The antigen recognized by Hy30 is found on greater than 95% of all bursal cells, on a population of thymus resident cells, and on blood, spleen, and marrow mononuclear cells. Immunohistological analysis of tissue sections revealed that the majority of the staining by Hy30 occurs in the B cell areas of the spleen and thymus. Flow cytometric analysis demonstrated that the antigen recognized by Hy30 is found on both immunoglobulin positive (Ig+) and negative (Ig-) cells. Analysis of a chicken rendered B cell deficient by treatment with cyclophosphamide at hatch confirmed this observation. In these birds, the medullary dendritic cells of the bursa and Ig- cells of the "B-cell areas" of the spleen and thymus expressed the Hy30 Ag. Analysis of several avian tumor lines did not suggest an obvious lineage for these Hy30+ Ig- cells. However, analysis of germinal centers suggested that these cells may be tissue macrophages or dendritic cells. This is consistent with previously reported histology of these "B-cell areas". Flow cytometric analysis of the antigen recognized by Hy30 demonstrates that it is distinct from the Bu-1 B-cell surface antigen and we designate it as Bu-2.