A disulfide-linked heterodimeric antigen from human B leukemia cells was detected by radioimmunoprecipitation and Western blot analysis using a monoclonal antibody (mAb). The mAb was generated against a cell membrane antigen preparation from human B prolymphocytic leukemia cells and found to define an extracellular epitope of the smaller component (beta chain) of a heterodimeric antigen on human B leukemia cells. The antigen from BALL-1, a human B leukemia cell line, and fresh (uncultured) B prolymphocytic leukemia cells was found to consist of a 44-49 kDa (alpha chain) and a 36-40 kDa (beta chain) component. An additional minor component of 34 kDa was detected in the reduced antigen from BALL-1. For chemical identification of the antigen, we isolated the antigen from a Triton X-100 lysate of BALL-1 by immunoaffinity chromatography using the mAb. Determination of the amino-terminal amino acid sequences of the alpha and beta chains unequivocally identified them as the human mb-1 and B29 proteins, respectively. The sequence analyses indicate the molecular heterogeneity of the mb-1 protein and also perhaps the heterogeneity of the B29 protein. We detected three forms of the mb-1 protein which share an identical amino-terminal amino acid sequence and probably two forms of the B29 protein which share an identical amino-terminal sequence. Our sequence data allowed us to establish the authentic amino-terminal amino acid sequence of the human B29 protein which is different from those proposed by others based on cDNA sequence analyses. Comparison of the amino-terminal sequences of the human mb-1 and B29 proteins with those of the mouse mb-1 and B29 proteins showed that the majority of the conserved amino acids in the mb-1 proteins are hydrophobic amino acids. Such conservation of hydrophobic amino acids is not observed in the amino termini of the human and mouse B29 proteins. A beta-tubulin-like protein was found to be co-purified with the mb-1-B29 antigen in the present study. In addition, we found that there is a strong amino acid sequence homology between the microtubule-binding domains of certain human microtubule-associated proteins and an intracellular segment of the human mb-1 protein.