A versatile genetic method for identifying and cloning Drosophila melanogaster genes affecting any recognizable phenotype is described. Strains are constructed in which the insertion of a single P transposable element has caused a new mutation, greatly simplifying the genetic and molecular analysis of the affected gene. Mutagenesis is initiated by crossing two strains, each of which contains a specially designed P element. One element (jumpstarter), encoding P element transposase, efficiently mobilizes the second nonautonomous transposon (mutator), whose structure facilitates selection and cloning of new insertion mutations. Random mutator transpositions are captured in individual stocks that no longer contain jumpstarter, where they remain stable. This method was used to construct 1300 single P element insertion stocks which were then screened for recessive mutations. A library of single-element insertion strains will allow the structure and function of Drosophila genes to be readily correlated, and should have many other applications in Drosophila molecular genetics.