In order to unravel the serine proteinase inhibition mechanism by cyclic thiolic compounds, the X-ray crystal structure of bovine alpha-chymotrypsin inhibited by 3-[2-(2-thiophencarboxythio)]-propanoyl-4-thioazolidin carboxylic acid (YS3025) has been studied by means of difference Fourier techniques and refined at 2.8 A resolution (R-factor = 0.174). The thiophencarbonyl moiety of YS3025 is covalently linked, in the acyl-enzyme complex, to Ser195 OG atom of bovine alpha-chymotrypsin, and located at the entrance of the proteinase primary specificity subsite (S1). The present data confirm previous hypotheses raised on the inhibition mechanism of serine proteinases, based on solution studies of human leukocyte elastase in the presence of the YS3025 parent compound 2-[3-thiophencarboxythio]-N-[dihydro-2(3H)- thiophenone-3-il]-propionamide (MR889).