Insertional mutagenesis with transposable P elements has greatly facilitated the identification and analysis of genes located throughout the 70% of the Drosophila melanogaster genome classified as euchromatin. In contrast, genetically marked P elements have only rarely been shown to transpose into heterochromatin. By carrying out single P element insertional mutagenesis under conditions where position-effect variegation was suppressed, we efficiently generated strains containing insertions at diverse sites within centromeric and Y-chromosome heterochromatin. The tendency of P elements to transpose locally was shown to operate within heterochromatin, and it further enhanced the recovery of heterochromatic insertions. Three of the insertions disrupted vital genes known to be present at low density in heterochromatin. Strains containing single P element insertions will greatly facilitate the structural and functional analysis of this poorly understood genomic component.