As a partial D antigen of the Rh blood group system, the D category IIIa phenotype occurs mainly in Blacks, but its molecular basis has not been defined. Here we describe studies of the D category D IIIa and VS+ red blood cells (RBC) from two unrelated probands by Southern blot, cDNA PCR, and nucleotide sequencing. Rh haplotyping by Sph I restriction fragment length polymorphisms indicated that the two probands carried Dce/dCe and Dce/DcE genotypes, respectively. Sequence analysis of Rh cDNAs showed that their erythroid cells expressed both D and CE transcripts. Nevertheless, the D transcripts were found to contain four nucleotide changes scattered in three exons: nt455 A-to-C (exon 3), nt602 C-to-G (exon 4), nt 654 C-to-G (exon 5), and nt667 T-to-G (exon 5). These variations resulted in the following amino acid substitutions characteristic of RhCE polypeptides: 152 Asn-to-Thr, 201 Thr-to-Arg, 218 Ile-to-Met, and 223 Phe-to-Val. The 152Thr and 223Val residues were predicted to reside in proximity to the third and fourth extracellular loops, respectively. Together, these results establish a correlation of the four amino acid changes in the RhD protein with the expression of D IIIa as a partial D antigen on the RBC membrane. Since the varied nucleotides identified in D IIIa all pre-exist in CE, they are likely to have originated from CE by templated micro-conversion event(s). The identification of a specific nt736 C-to-G transversion in CE in the two probands suggests that 245Val may involve the expression of VS antigen. Am.