The interaction between five N-terminally truncated forms of chicken cystatin (starting at Leu-7, Leu-8, Gly-9, Ala-10 and Asp-15) and the cysteine proteinases papain and actinidin was studied by spectroscopic, kinetic and equilibrium methods. The u.v. absorption, near-u.v. c.d. and fluorescence emission difference spectra for the interactions with papain were all similar to the corresponding spectra for intact cystatin. The second-order association rate constants at 25 degrees C, pH 7.4, I 0.15, for the binding of the truncated forms to papain varied about 2-fold, from 6 x 10(6) to 1.5 x 10(7) M-1.s-1, and were comparable to the value of 9.9 x 10(6) M-1.s-1 for intact cystatin. In contrast, the rate constants for the dissociation of the complexes with papain increased markedly with increasing extent of truncation, from 7.5 x 10(-6)s-1 for Leu7 cystatin (a truncated form of cystatin having Leu-7 as its N-terminal amino acid) to 1.6s-1 for Ala10-cystatin, whereas the dissociation rate constants for the latter form and Asp15-cystatin were similar. Consequently, the binding affinities between the truncated cystatins and papain decreased in an analogous manner, as was also shown for the interaction with actinidin by equilibrium measurements. Studies of the binding of the truncated cystatins to inactivated papains indicated that small substituents on the active-site cysteine of the enzyme can be accommodated in the complex without any loss of affinity when the N-terminal segment of the inhibitor is removed. Taken together, the results suggest that in the N-terminal region of chicken cystatin only residues preceding Ala-10 participate in the interaction with proteinases. Of these residues, Leu-7 and Leu-8 together account for about two-thirds of the unitary free energy of binding contributed by the N-terminal region, the relative importance of the two residues being dependent on the target proteinase. Both Gly-9 and residues N-terminal of Leu-7 further stabilize the interaction but contribute substantially smaller binding energies than do the two leucine residues.