The human glycophorin (HGp) loci that define the red blood cell surface antigens of the MNSs blood group system exhibit considerable allelic variation. Previous studies have identified gene conversion events involving HGpA(alpha) and HGpB(delta) that produced delta-alpha-delta hybrid genes which differ in the location of breakpoints. This report presents the molecular analysis of HGpMilX, the first example of a reverse alpha-delta-alpha hybrid gene that specifies a newly described phenotype of the Miltenberger complex. A novel restriction fragment unique to the HGpMilX gene was detected by Southern blot hybridization. The structure of the genomic region encoding the entire extracellular domain of the MilX protein was determined. Nucleotide sequencing of amplified genomic DNA showed that a silent segment of the HGpB(delta) gene had been transposed to replace the internal part of exon III in the HGpA(alpha) gene, thereby resulting in the formation of the MilX allele with an alpha-delta-alpha configuration. The proximal alpha-delta breakpoint was found to be flanked by a direct repeat of the acceptor splice site, whereas the distal delta-alpha breakpoint was localized to a palindromic region. This DNA rearrangement, with a minimal transfer of 16 templated nucleotides and a single mutation of untemplated adenyl nucleotide, not only created two intraexon hybrid junctions but transactivated the expression of a new stretch of amino acid residues in the MilX protein. Such a segment replacement may have occurred through the directional transfer from one duplex to the other via the mechanism of gene conversion. The occurrence of HGpMilX as another hybrid derived from parts of parent genes underlines the role of the recombinational "hotspot" in the generation of allelic diversity in the glycophorin family.