1. The kinetic parameters of 25 peptidyl-p-nitroanilide substrates were investigated with subtilisin Carlsberg as model enzyme. 2. For a series of 12 substrates, the contribution of various side chains to the affinity constant was computed by regression analysis. From these contributions the sequence of a new and better substrate, N-benzyloxycarbonyl-arginyl-norleucyl-norleucyl-p-nitroanilide (Z-Arg-Nle-Nle-Nan) was predicted. The compound was synthesized and assayed. Its calculated 1/Km value, 43.5 mM-1, was in a good agreement with the value of 40.0 mM-1 that was determined experimentally. 3. On expanding the series to 19 substrates, it was found that the productivity of enzyme-substrate binding is influenced primarily by those subsites which have a significantly greater contribution to the affinity constants than others. 4. The additivity principle applied reasonably well for the contribution of individual side chains to the kinetic parameters. This fact suggests that regression analysis can be used for the prediction of the amino acid sequence of better substrates than those already tested, probably not only for subtilisin but also for other proteolytic enzymes.