BACKGROUND: The GP.Hop (Mi.IV) phenotype expresses the MNS low‐incidence antigens Mur, Hop, TSEN, MINY, and MUT. Because serologically similar MNS phenotypes expressing some or all of these antigens were shown to be carried by hybrid GP(B‐A‐B) proteins, it was proposed that a similar protein would be found for GP.Hop. The identification of a second GP.Hop propositus (ES) initiated a study to determine the molecular basis of this phenotype.STUDY DESIGN AND METHODS: Serologic tests and immunoblotting analysis with glycophorin‐specific antibodies were performed. GYPB, the gene encoding the GPB protein, was cloned and sequenced after reverse transcription PCR amplification of total RNA isolated from ES. GYPB‐specific primers encompassing GYPB pseudoexon 3, intron 3, and exon 4 were also used to clone and sequence genomic DNA from ES and MH, the original GP.Hop proband.RESULTS: Serologic and immunochemical data confirmed that ES's RBCs carried antigens associated with the GP.Hop phenotype. Sequencing of ES's cDNA demonstrated the presence of genes predicted to encode s‐specific GPB and an S‐specific GP(B‐A‐B) hybrid in which the 3′ end of GYPB pseudoexon 3 had been replaced by a short nucleotide sequence from exon 3 of the GPA gene (GYPA). The hybrid nucleotide sequence contained sequence motifs previously shown to be required for the expression of the Mur, Hop, TSEN, MINY, and MUT, which is consistent with their presence as detected serologically. Genomic DNA analysis found that the crossover point in GYPB pseudoexon 3 was identical in ES and MH.CONCLUSIONS: The GP.Hop phenotype is produced by a hybrid GP(B‐A‐B) protein caused by a DNA insertion of GYPA into GYPB. The composition of the hybrid protein is GPB(1‐26)‐GPψB(27‐50)‐GPA(51‐58)‐GPBS(59‐103).