Human cystatin D is a novel member of the cystatin superfamily of cysteine proteinase inhibitors present in saliva and tears. Two alleles of the cystatin D gene (CST5), encoding protein variants with either Cys or Arg as residue 26 in their 122-residue polypeptide chains, are present in the population. Expression of the two alleles was investigated by immunochemical analyses of the secreted cystatin D in saliva from individuals homozygous for each of the two alleles, with results demonstrating that both are expressed at similar levels. The inhibitory characteristics of the two cystatin D variants were studied, by determination of dissociation equilibrium constants (Ki) for their complexes with papain and with the mammalian cysteine proteinases, cathepsins B, H, L, and S. The results demonstrate that 1) cystatin D has a characteristic inhibition profile since it does not inhibit cathepsin B (Ki > 1 microM), and when compared to cystatin C and all other known cystatins it is a much poorer inhibitor of cathepsin L (mean Ki 25 nM) but binds cathepsin H and S relatively tightly (mean Ki values of 8.5 and 0.24 nM, respectively); and 2) the inhibitory activities of the two cystatin D variants are not significantly different, demonstrating that the presence of an extra cysteine residue in the cystatin D molecule affects neither the stability nor the functional activity of the inhibitor, thus explaining the widespread distribution of the Cys26-cystatin D encoding allele in the population. The inhibitory properties displayed by cystatin D suggest that it has a function in saliva as inhibitor of either endogenous or exogenous enzymes with cathepsin S- or H-like properties.