We have characterized an unusual family with two differentandrogen receptor(AR) gene deletions, in which we propose a novel mechanism of deletion formation has occurred. Affected individuals have the X-linked disorder androgen insensitivity syndrome, and we previously showed that different family members have deletions of different exons of theARgene. We have now fully sequenced the deletions from affected individuals, and confirmed the presence of different deletions in different affected family members. Most affected and heterozygote individuals have a 4,430-bp deletion of exon 5 that occurred between repeated GTGGCAT motifs in introns 4 and 5. One affected hemizygous individual has a 4,033-bp deletion of exons 6 and 7 that occurred between repeated CCTC motifs in introns 5 and 7. The intron 5 breakpoint junctions of the two deletions are only 11 bp apart. Surprisingly, the maternal grandmother of the original index case was found to be mosaic for both deletional events, as well as having the normalARgene. Karyotyping ruled out 47,XXX trisomy, indicating triple mosaicism for the two different deletedARalleles and a normalARallele. This triple mosaicism must have occurred early in embryonic development, as both deletions were passed on to different children. Based on these findings, we propose a novel mechanism of deletion formation. We suggest that duringARgene replication, a double strand DNA break occurred in intron 5, and that a variant of replication slippage occurred on both newly synthesized strands between the repeat motifs of microhomology, leading to the formation of the two differentARgene deletions. Hum Mutat 27(5), 483-489, 2006. © 2006 Wiley-Liss, Inc.