Various mutations in E. coli alkaline phosphatase gene were obtained by oligonucleotide-directed mutagenesis. They result in amino acid substitutions in the signal peptide cleavage site [Val for Ala(-1)] and in the N terminus of mature polypeptide chain: Ala for Arg(+1) and Gln for Glu(+4); Gln for Glu(+4). Enzyme activity was observed in all E. coli strains transformed by plasmids with cloned mutant genes. In addition, an amber mutation was introduced into the Arg(+1) position, and the synthesis of mutant alkaline phosphatase was shown in E. coli strains containing suppressor tRNAs specific for Ser, Gln, Tyr, Leu, Ala, Glu, Phe, Gly, His, Pro, and Cys.