Cathepsin C was purified from human spleen by a rapid procedure, which included homogenization, ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and finally affinity chromatography on chicken cystatin-Sepharose. The interaction between cathepsin C and chicken cystatin was further characterized. It was found to be accompanied by a maximum decrease in fluorescence emission intensity at 330 nm. Fluorescence titration showed that human cathepsin C can bind four chicken cystatin molecules. The 4:1 binding stoichiometry was confirmed by titration monitored by the loss of enzyme activity. A non-competitive-competitive type of inhibition was determined from a double-reciprocal Lineweaver-Burk plot with a K i value of 0.22 nM for the non-competitive inhibition.