Mutation of the alpha-actinin gene in Dictyostelium has been achieved by transforming cells with the Dictyostelium transformation vector pDNeoII containing a 1.2 kb fragment of the alpha-actinin gene. Transformants deficient in alpha-actinin, an actin-binding protein, produced an altered mRNA that lacked the 3' portion of the coding region. The defect in alpha-actinin production was not due to integration of the vector within the gene, but was apparently caused by errors produced during homologous recombination between the introduced alpha-actinin sequence and its complementary sequence in the coding region of the endogenous gene.