Genetic analysis of protein targeting in Escherichia coli has been facilitated by the use of lacZ gene fusions. Strains which contain a fusion between a gene specifying a noncytoplasmic protein and lacZ exhibit novel phenotypes that can be exploited to obtain export-defective mutations. Analysis of these mutations has demonstrated the importance of the signal sequence and provided some insight into the functions performed by this complex intragenic export signal. In addition, genes that specify components of the cellular protein export machinery have been identified. Three of these genes, secA, secB, and prlA/secY have been studied in some detail and results indicate that the protein products function in the translocation of exported proteins across the cytoplasmic membrane.