While investigating the copy number of minichromosome Dp(1;f)1187 sequences in the polyploid chromosomes of ovarian nurse and follicle cells of Drosophila melanogaster we discovered that restriction fragments spanning the euchromatic-heterochromatic junction of the chromosome and extending into peri-centromeric sequences had the unusual property of being selectively resistant to transfer out of agarose gels during Southern blotting, leading to systematic reductions in Dp1187-specific hybridization signals. This property originated from the peri-centromeric sequences contained on the junction fragments and was persistently associated with Dp1187 DNA, despite attempts to ameliorate the effect by altering experimental protocols. Transfer inhibition was unlikely to be caused by an inherent physical property of repetitive DNA sequences since, in contrast to genomic DNA, cloned restriction fragments spanning the euchromatic-heterochromatic junction and containing repetitive sequences transferred normally. Finally, the degree of inhibition could be suppressed by the addition of a Y chromosome to the genotype. On the basis of these observations and the fact that peri-centromeric regions of most eukaryotic chromosomes are associated with cytologically and genetically defined heterochromatin, we propose that peri-centromeric sequences of Dp1187 that are incorporated into heterochromatin in vivo retain some component of heterochromatic structure during DNA isolation, perhaps a tightly bound protein or DNA modification, which subsequently causes the unorthodox properties observed in vitro.