The pentapeptide pepstatin was found to inhibit the ability of rat spleen pseudorenin to form angiotensin I from tetradecapeptide renin substrate. Dixon and Webb plots showed that this inhibition was noncompetitive in nature. Lineweaver-Burk analysis also showed noncompetitive inhibition. Ki values determined by the three graphical methods ranged from 1.8 to 3.8 × 10-10M. The Km for pseudorenin was determined to be between 0.82 and 1.23 μM. The concentration of enzyme used was estimated to be 3.1 × 10-10M. Pepstatin should prove useful in the future for the analysis and purification of pseudorenin.