An automated method using 2,2'-dithiodipyridine (2-PDS) as chromophore for determination of whole-blood cholinesterase activity was developed. Assay procedures, optimal concentrations of chromophore and substrate, detection limit, precision, backgrounds, and sensitivity of the method were compared with those of an earlier automated method based on the Ellman method and using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as chromophore. The new method has the advantages of automation (resulting in increase throughput rate and decrease in amount of reagents used) and good precision and sensitivity. Sample dilutions also are reduced in the new method because hemoglobin interference is less.