In pre-B cells, mu chains are expressed in association with "surrogate' L chains encoded by the lambda 5 and Vpre-B1 genes. In addition to their association with lambda 5 and Vpre-B1, mu chains in pre-B cells are associated with the products of the Vpre-B3 gene (formerly designated 8HS-20), which display a distinct association with mu chains and biochemical properties in terms of mol. wt, pI value and glycosylation. However, the mechanism of the generation of Vpre-B3 isoforms has been unknown. The present study indicates that the Vpre-B3 gene transcript underwent alternative RNA processing in normal B cells, in a pre-B cell lymphoma and in a mature B cell lymphoma, WEHI 231, that was transfected with the Vpre-B3 genomic clone. Vpre-B3 isoforms were expressed in a WEHI 231 cell line transfected with the Vpre-B3 genomic clone, comparable in biochemical nature to those expressed in a pre-B cell lymphoma. In contrast, expression of one of the isoforms was missing in a cell line transfected with the Vpre-B3 cDNA clone. These results suggest that Vpre-B3 isoforms with distinct biochemical characteristics are derived from alternatively processed Vpre-B3 mRNA.