A number of peptide-4-nitroanilide substrates containing proline within the peptide chain have been synthesized and subjected to chymotryptic hydrolysis. Values of kcat and Km have been obtained from measurements at pH 7.8 and 25.0 degrees C. Kinetic studies at high enzyme concentrations up to 6.0 X 10(-4) mol X 1(-1) have allowed the evaluation of the conformational specificity of chymotrypsin due to the observation of various kinetic phases during the time-course of the reaction. When proline occupies the P2 position within the peptide chain, it is shown that the enzyme cleaves only the trans isomer of the substrate. The conformational specificity has also been studied for proline in P4 and P5 positions of the substrate. In some cases, an enzyme-catalyzed hydrolysis of the cis isomer was detected. From the amplitude ratios and the rate constants of the kinetic phases, information about the structural dependency of the cis/trans interconversion could be obtained. Charged residues N-terminal to the isomeric bond are of little influence on either cis/trans ratio or the rate of cis to trans interconversion. Extending the peptide chain N-terminal to the isomeric bond by alanine decreases to a low extent the cis content and increases the rate constant of the trans isomer formation.