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Regulation of gene expression is a highly complex process that requires the concerted action of many proteins, including sequence-specific transcription factors, cofactors, and chromatin proteins. In higher eukaryotes, the interplay between these proteins and their interactions with the genome still is poorly understood. We systematically mapped the in vivo binding sites of seven transcription factors with diverse physiological functions, five cofactors, and two heterochromatin proteins at [approximately equal to] 1-kb resolution in a 2.9 Mb region of the Drosophila melanogaster genome. Surprisingly, all tested transcription factors and cofactors show strongly overlapping localization patterns, and the genome contains many 'hotspots' that are targeted by all of these proteins. Several control experiments show that the strong overlap is not an artifact of the techniques used. Colocalization hotspots are 1-5 kb in size, spaced on average by [approximately equal to] 50 kb, and preferentially located in regions of active transcription. We provide evidence that protein-protein interactions play a role in the hotspot association of some transcription factors. Colocalization hotspots constitute a previously uncharacterized type of feature in the genome of Drosophila, and our results provide insights into the general targeting mechanisms of transcription regulators in a higher eukaryote. chromatin profiling | DamID | tiling array | transcriptional regulator