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Summary 1. Attempts were made to develop an automated method which could perform quantitative measurements of total serum pseudocholinesterase (E.C. 3.1.1.8.) activity and dibucaine number by use of the Hestrin reaction. 2. Unusual patterns of inhibition were observed with dibucaine; some inhibited sera gave absorbance values which were higher than the absorbance of the substrate itself. This is due to the fact that certain sera contain “ester-like” substances which dialyze and thereafter react with hydroxylamine to produce a complex absorbing at the same wavelength as the acylhydroxamate ferric complex. 3. Application of the Hestrin reaction for the determination of total serum pseudocholinesterase activity and dibucaine number with an automated system equipped with a dialyzer therefore requires corrections for serum blank values. Consequently, the procedure does not appear to be practical for large scale determinations of these parameters.