The HLA-DNA gene of the human major histocompatibility complex (MHC) produces RNA transcripts of unusual size compared to the 1.3 kilobase (kb) transcripts of other class II genes. Several cDNA clones derived from HLA-DNA transcripts were isolated, including a full-length 3.5 kb cDNA named DN1. Sequence analysis of DN1 demonstrated that the 3.5 kb transcript is fully spliced, contains a long 3' untranslated region, and encodes an apparently normal class II alpha chain. Two other cDNA clones, DN2 and DN7, revealed an alternative splice that resulted from the use of a splice acceptor upstream of the second exon encoding the alpha 1 domain. The translation product of this alternative transcript would share the signal sequence of the DN alpha chain but would otherwise be unrelated. Removal of the signal sequence would yield a highly basic peptide of 42 amino acids. RNA was prepared by in vitro transcription of DN1 and DN2, and was used to direct protein synthesis in a rabbit reticulocyte system. The product of DN1 had an apparent relative mass of 25,000, whereas the product of DN2 was a peptide with a relative mass of about 6000. The alternatively spliced RNA represents about 1/10 of the transcripts from the HLA-DNA gene in the cells tested, which included transformed B-cell lines as well as normal blood cells and thymus cells.