One possible reason why regeneration remains enigmatic is that the dominant organisms used for studying regeneration are not amenable to genetic approaches. We mutagenized zebrafish and screened for temperature-sensitive defects in adult fin regeneration. The nightcap mutant showed a defect in fin regeneration that was first apparent at the onset of regenerative outgrowth. Positional cloning revealed that nightcap encodes the zebrafish orthologue of mps1, a kinase required for the mitotic checkpoint. mps1 expression was specifically induced in the proximal regeneration blastema, a group of cells that normally proliferate intensely during outgrowth. The nightcap mutation caused severe defects in these cells. However, msxb-expressing blastemal cells immediately distal to this proliferative region did not induce mps1 and were retained in mutants. These results indicate that the proximal blastema comprises an essential subpopulation of the fin regenerate defined by the induction and function of Mps1. Furthermore, we show that molecular mechanisms of complex tissue regeneration can now be dissected using zebrafish genetics.