CD46 (membrane cofactor protein) is a human cell surface glycoprotein with cofactor activity for factor I-mediated cleavage of complement components C3b and C4b. The CD46 protein from normal lymphocytes resolves on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two major bands of 66 and 56 kDa. CD46 cDNA encodes four extracellular short consensus repeat domains, a Ser/Thr/Pro (STP)-rich region, a transmembrane region and a cytoplasmic tail. We now show that exquisite control of mRNA splicing is responsible for the heterogeneous expression of CD46 isoforms. Differential splicing of 5 exons generates at least 14 CD46 mRNA variants whose expression is stringently regulated by allelic, tissue-specific and malignancy-related factors, as: (a) leukemic cells and Epstein-Barr virus-transformed B cells preferentially incorporate the first of three STP exons (exon 7) into mRNA, and produce a larger CD46 isoform of 74 kDa, (b) an allelic difference in the proportion of 66- and 56-kDa CD46 isoforms on lymphocytes corresponds to the preferential inclusion or exclusion of the second STP exon (exon 8), (c) the third STP exon (exon 9) is specifically deleted in some placentae, (d) spermatozoa delete both exons 12 and 13, encoding a shorter transmembrane region and a unique cytoplasmic tail and (e) all tissues tested differentially splice exon 13, resulting in two alternative cytoplasmic tails. The distribution of the 14 alternatively spliced RNA transcripts correlated with the presence of protein isoforms of the predicted size, indicating that alternative splicing leads to heterogeneity of CD46 glycoproteins.