Enhancer trapping allows the detection of genomic transcriptional enhancers and provides a rich source of cell- and region-specific markers in Drosophila. We report here the development of a P-element-based enhancer-trap vector with a nuclear red fluorescent protein (DsRed) reporter for enhancer detection. We demonstrate that this vector can be used for a standard enhancer-trap screen as well as for targeted replacement of previously characterized P-element insertions. We isolated DsRed insertion strains of hedgehog, patched, apterous, teashirt, and dachshund that label specific regions of imaginal discs. The time required for red fluorescence maturation in the eye imaginal disc was estimated to be 22.5 hr. The DsRed markers can be combined with green fluorescent protein markers for double labeling of living Drosophila tissues.