Site directed mutagenesis was used to investigate the role of Ser-143 in enzyme activity and as a point for attack by cyanide or L-cysteine, two irreversible inhibitors of histidine ammonia-lyase (histidase). Two mutant proteins, a S143A substitution and an A142S-S143A transposition, were made. Both mutant histidases completely lost all enzymatic activity. Western blots with anti-histidase antibodies revealed that the mutant proteins were being expressed at a level equal to that of the wild-type protein. The purified mutant proteins could not incorporate [14C]cyanide nor could they generate the UV-absorbing species normally observed when L-cysteine modifies wild-type histidase. These results support the hypothesis that Ser-143 is the binding site for an as yet unidentified histidase cofactor.