The Rh blood group antigens are carried by two distinct but homologous membrane proteins encoded by two closely related genes,RHCEandRHD.Rh50 glycoprotein is the membrane protein tightly associated with Rh polypeptides and is critical for expression of Rh antigens. The amino acid sequence and predicted membrane topology of Rh50 glycoprotein are significantly homologous with those of the Rh proteins. Northern blot analysis of leukemic cell lines showed that expression ofRH50gene is restricted to cells with erythroid features. HEL and K562 cells showed a transcription levels ratio of 1 to 9.9 for Rh50, and 12.3 to 1 for Rh. The nucleotide sequence of 5' flanking region ofRH50gene and functional promoter assays also supported the erythroid-specific regulation of the gene, whereas the sequence had lower homology with the promoter sequence ofRHgenes. Seven GATAs, nine E-boxes, two CACCCs, one YY1, and one October motif were identified in the 1868bp 5' flanking sequence. The core promoter ofRH50gene was located within 68bp length from the translation start position, which included an inverse GATA motif, although obvious motifs for Sp1 or erythroid Kruppel-like factor were lacking. The inverse GATA motif was the target sequence of GATA-1 protein, and disruption of the motif abolished the transactivating activity of erythroid cells. These studies confirm the erythroid-specific expression of Rh antigens, but suggest distinct regulatory mechanisms forRHvsRH50genes.