The ATP-dependent casein hydrolysis by protease Ti (ClpAP) has been shown to be inhibited by sulfhydryl blocking agents, such as N-ethylmaleimide (NEM), when preincubated with ClpA but not with ClpP. To define the role of three Cys residues in ClpA, site-directed mutagenesis was performed to substitute each of them with Ser or Ala. None of the mutations showed any effect on the ATPase activity of ClpA or its ability to support the casein degradation by ClpP. However, NEM could no longer block the ability of ClpA/C47S or ClpA/C47A in supporting the ClpP-mediated proteolysis, unlike that of ClpA, ClpA/C203S, or ClpA/C243S. Furthermore, in the presence of NEM, casein could stimulate the ATPase activities of ClpA/C47S and ClpA/C47A and protect from their degradation by ClpP, but not of the other ClpA proteins. These results suggest that the inhibitory effect of NEM is due to prevention of the interaction of ClpA with casein by introduction of a bulky alkyl group to Cys47, but not linked to the catalytic function of the ATPase.