Conditional targeted cell ablation in zebrafish would greatly expand the utility of this genetic model system in developmental and regeneration studies, given its extensive regenerative capabilities. Here, we show that, by combining chemical and genetic tools, one can ablate cells in a temporal‐ and spatial‐specific manner in zebrafish larvae. For this purpose, we used the bacterial Nitroreductase (NTR) enzyme to convert the prodrug Metronidazole (Mtz) into a cytotoxic DNA cross‐linking agent. To investigate the efficiency of this system, we targeted three different cell lineages in the heart, pancreas, and liver. Expression of the fusion protein Cyan Fluorescent Protein–NTR (CFP‐NTR) under control of tissue‐specific promoters allowed us to induce the death of cardiomyocytes, pancreatic β‐cells, and hepatocytes at specific times. Moreover, we have observed that Mtz can be efficiently washed away and that, upon Mtz withdrawal, the profoundly affected tissue can quickly recover. These findings show that the NTR/Mtz system is effective for temporally and spatially controlled cell ablation in zebrafish, thereby constituting a most promising genetic tool to analyze tissue interactions as well as the mechanisms underlying regeneration. Developmental Dynamics 236:1025–1035, 2007. © 2007 Wiley‐Liss, Inc.