Male or female DNA of Drosophila hydei was sheared by sonication, denatured, reannealed to different C0t-values and fractionated by hydroxyapatite. The highly repetitive, moderately repetitive and unique fractions of female DNA were denatured again and coupled via diazotization or cyanogen bromide to macroporous Sephacryl S-500. Enrichment of Y-chromosomal sequences was achieved by cycling each of the different fractions of male DNA under optimized hybridization conditions over a column with a manifold excess of immobilized female DNA of the corresponding complexity. Thereby, Y-chromosomal sequences of D. hydei could be enriched about 100-fold for highly and moderately repetitive DNA and about 10-fold for unique DNA. When a library of male D. hydei DNA was screened with Y-enriched highly repetitive DNA, more than 98% of all hybridizing phages contained inserts of repetitive Y-chromosomal DNA of at least four different sequence families.