We report here on the order of three DNA markers, C7, B24, and L1, based on the arrangement of their fluorescently labeled hybridization sites in interphase cell nuclei. The three markers map distal to the Duchenne muscular dystrophy (DMD), glycerol kinase deficiency (GKD), and adrenal hypoplasia (AHC) loci on human chromosome Xp21.3. Their order has been a matter of controversy. In interphase chromatin, B24 maps between C7 and L1. We estimate from interphase distance that C7 and L1 are 300-500 kb apart. When the three markers are hybridized to interphase cells of Nijmegen1, a patient with DMD, GKD, and AHC, only C7 appears to be deleted, rather than both C7 and L1, as had been reported elsewhere. C7 is also the only one of the three markers deleted in several other DMD patients studied by others. The deletion results indicate that C7 is the most proximal of the three markers and allow the trio of ordered probes to be oriented on the chromosome: cen-C7(DXS28)-B24(DXS67)-L1(DXS68)-tel.