The acetylcholinesterase (EC 3.1.1.7) in 50 microL of a 61-fold dilution of erythrocytes in water hydrolyzes acetylcholine during a timed 20-min reaction at 37 degrees C. The resulting choline is measured by use of choline oxidase coupled to peroxidase, with phenol and aminoantipyrene to give a pink product that absorbs maximally at 500 nm. For calibration, a choline iodide standard is included in each batch of up to 19 samples. Accuracy was assessed by using specific inhibitors and measuring choline in the presence of excess erythrocyte solution. The standard curve for the assay is linear to threefold the normal enzyme activity. Between-batch precision was 0.40 kU/L at a mean of 11.5 kU/L (CV 3.5%), and comparison with an acetylthiocholine procedure (x) gave a good correlation: y = 1.02x - 0.27 kU/L (r = 0.991). Long-term precision (10 months), assessed from three sets of assays of samples from 17 individuals, was 0.71 kU/L at a mean of 11.7 kU/L (CV 6.1%).