Treatment of small cells derived from the basal layer of bovine esophageal epithelium, with Triton X-100, urea and sonication resulted in a nuclear residue that was used as an immunogen for generation of monoclonal antibodies directed against nuclear components. One such antibody, designated W1, was found to label the nuclei of all cells examined. In interphase cells, the target antigen of antibody W1 was diffusely distributed in the nucleus. During metaphase, however, the W1 antigen formed prominent crescents at the poles of the mitotic spindle, diminished gradually in anaphase, and finally redistributed into the regenerating daughter nuclei. Western blotting with antibody W1 yielded a prominent polypeptide of M(r) approximately 230,000. The amino acid sequence, deduced from the nucleotide sequence of several overlapping cDNA clones that span the entire coding region, revealed that the W1 polypeptide was identical to the Nuclear Mitotic Apparatus (NuMA) protein, with a long alpha-helical central core flanked by two nonhelical domains. Interestingly, most cDNA sequences were identical to each other, except for six sequence blocks which were either inserted or deleted in individual cDNA clones. Analysis of the cDNA sequences of various clones, coupled with polymerase chain reaction amplification of cellular mRNA and genomic Southern blotting with region-specific probes, all indicated that multiple mRNA species were present in U-251 human glioma cells, derived from alternative splicing of the RNA transcript from a single NuMA/W1 gene. Besides the predominant form of the mRNA giving rise to the polypeptide of M(r) approximately 230,000, two other forms of mRNA, which arise as a result of alternative splicing and which use different translation termination codons, may yield lower molecular weight polypeptide products. Consistent with this notion, polypeptides of M(r) approximately 195,000 and approximately 194,000 have been observed in this and other studies on the NuMA/W1 protein. These data suggest that multiple isoforms of the NuMA polypeptides generated by alternative mRNA splicing may play some important functions which remain to be characterized.