A method for the integration of linear DNA into the Dictyostelium genome is described. Restriction enzyme-mediated integration, or REMI, involves the transformation of cells with a mixture of plasmid DNA, linearized with a restriction enzyme, along with a restriction enzyme that is capable of generating compatible cohesive ends in the genome. The enzyme stimulates integration of the DNA into cognate restriction sites in the chromosomes, usually as a single-copy insertion event and with little collateral damage to the genome. REMI has proven useful for genetic screens and for placing genetic and molecular markers at particular points in the genome. Over the past 15 yr, REMI has been used to identify hundreds of interesting genes based on their mutant phenotypes.