It was shown that at least four genes are specifically responsible for arylsulfatase synthesis in Klebsiella aerogenes. Mutations at chromosome site atsA result in enzymatically inactive arylsulfatase. Mutants showing constitutive synthesis of arylsulfatase (atsR) were isolated by using inorganic sulfate or cysteine as the sulfur source. Another mutation in which repression of arylsulfatase by inorganic sulfate or cysteine could not be relieved by tyramine was determined by genetic analysis to be on the tyramine oxidase gene (tyn). This site was distinguished from the atsC mutation site, which is probably concerned with the action or synthesis of corepressors of arylsulfatase synthesis. Genetic analysis with transducing phage PW52 showed that the order of mutation sites was atsC-atsR-atsA-tynA-tynB. On the basis of these results and previous physiological findings, we propose a new model for regulation of arylsulfatase synthesis.