MicroRNAs (miRNAs) are noncoding RNA molecules that have come to attract considerable interest for their roles in animal and plant development and disease. One means to study miRNA function in animal development is to create mutations. Use of gene-targeting strategies based on ends-out homologous recombination is a useful approach to produce mutations of desired structure, and is gaining popularity for producing miRNA knockouts. Here we present a detailed protocol for miRNA gene targeting and for their subsequent molecular characterization as well as confirmation by rescue. The descriptions of a series of modified vectors designed to facilitate the analysis of miRNA function are included, and a method to manipulate the mutant genome using recombinase-mediated cassette exchange.