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In Escherichia coli three major alkaline phosphatase isozymes are formed by molecular conversions depending on physiological conditions. A chromosomal gene, iap, is responsible for alkaline phosphatase isozyme conversion and is assumed to code for a proteolytic enzyme removing the arginine residue(s) from the N-terminal position of alkaline phosphatase subunits. A chromosomal fragment which complemented the Iap- phenotype was cloned into pBR322 by a shotgun method. Transducing phage lambda iap was constructed in vitro from the chromosomal fragment containing the iap gene and lambda tna DNA. The integration site of the phage on chromosome was identified as the iap locus by P1 transduction, which meant that the cloned chromosomal DNA contained authentic iap gene. The restriction map of the hybrid plasmid was constructed. Based upon this information, several iap deletion plasmids as well as smaller iap+ plasmids were constructed. Analysis of the phenotypes conferred by these plasmids enabled us to locate iap gene within a 2-kb segment of the cloned DNA. The cells carrying the iap+ plasmid showed very efficient isozyme conversion even in medium containing arginine, an inhibitor for the isozyme conversion. This indicates overproduction of the iap gene product.