Dithiothreitol (Cleland's reagent) is widely used as a sulfhydryl protective reagent in biochemical systems in vitro. For example, dithiothreitol has been used to achieve maximal rates of enzyme activity for protein phosphokinase reactions (1-4) as well as for phosphoprotein phosphatese assays (1,5). Meisler and Langan (5) have utilized 32P-labeled histone phosphoprotein as a substrate to examine the protein phosphatase activity of a rat liver cytosol enzyme preparation. However, if one is dealing with a phosphoprotein substrate which may not be labeled with 32P, it would be desirable to measure the phosphatase activity using a sensitive chemical analysis, e.g., the method of Berenblum and Chain (6) as modified by Martin and Doty (7). We have been interested in examining the protein phosphatase activity associated with prostatic chromatin and the androgenic influences thereupon, using nonhistone and histone phosphoproteins and phosvitin as substrates (Ahmed and Davis, unpublished data). In designing these experiments, 1-3 mm dithiothreitol was added in the reaction medium; this subsequently resulted in interference of Pi analysis using the Berenblum and Chain procedure (6,7). We have, therefore, systematically examined the conditions which may be used to assay Pi when dithiothreitol is present in the sample. The following report deseribes these observations.