One of the most powerful methods available to assign function to a gene is to inactivate or knockout the gene. Recently, we described the first target-selected knockout in zebrafish. Here, we report on the further improvements of this procedure, resulting in a highly efficient and easy method to do target-selected mutagenesis in zebrafish. A library of 4608 ENU-mutagenized F₁ animals was generated and kept as a living stock. The DNA of these animals was screened for mutations in 16 genes by use of CEL-I-mediated heteroduplex cleavage (TILLING) and subsequent resequencing. In total, 255 mutations were identified, of which 14 resulted in a premature stop codon, 7 in a splice donor/acceptor site mutation, and 119 in an amino acid change. By this method, we potentially knocked out 13 different genes in a few months time. Furthermore, we show that TILLING can be used to detect the full spectrum of ENU-induced mutations in a vertebrate genome with the presence of many naturally occurring polymorphisms.