A mutant producing very high levels of the acetamidase enzyme encoded by the amdS gene has been isolated in a strain containing the amdA7 mutation, which itself causes high levels of this enzyme. Genetic analysis has shown that this mutation, designated amdI66, is adjacent to the amdS gene and is cis-dominant in its effect. The amdI66 mutation has little effect on amdS expression when present in strains not containing the amdA7 mutation. Two other amdA mutations investigated also interact with the amdI66 mutation to result in high acetamidase levels. No interaction between amdI66 and any of the other putative regulatory genes affecting amdS expression has been observed. The amdI66 mutation has been located by fine structure mapping at the extreme end of the controlling region, which has previously been defined by genetic mapping (HYNES 1979). Analysis of this region has been extended by mapping new mutations resulting in loss of amdS expression. One of these defines the most extreme site capable of mutation to loss of gene function found so far.