We generated a ROSA26-eGFP-DTA mouse line by introducing an eGFP-DTA (enhanced green fluorescent protein -- diphtheria toxin fragment A) cassette into the ROSA26 locus by homologous recombination in ES cells. This mouse expresses eGFP ubiquitously, but DTA expression is prevented by the presence of eGFP, a Neo cassette, and a strong transcriptional stop sequence. Mice carrying this construct are normal and fertile, indicating the absence of DTA expression. However, upon Cre-mediated excision of the floxed region DTA expression is activated, resulting in the specific ablation of Cre-expressing cells. As an example of this approach, we ablated Nkx2.5 and Wnt1-expressing cells by using the Nkx2.5-Cre and Wnt1-Cre mouse lines, respectively. We observed loss of the precise tissues in which Nkx2.5 and Wnt1 are expressed. Apart from being a general GFP reporter, the ROSA26-GFP-DTA mouse line should provide a useful resource for genetic ablation of specific groups of cells.