We report a fully automated method for determining dibucaine number (DN) in a single-run procedure involving Dimension cholinesterase (CHE) Flex pseudo-(P)CHE reagents. The method was developed and optimized with the "open channels" and "kinetic" software facilities of the Dimension-ES instrument, where the DN is calculated automatically by an algorithm from the ratio of the uninhibited and inhibited rates, measured bichromatically, from a single analysis. The protocol was satisfactorily assessed for substrate depletion, linearity, reagent stability. and the effects of different dibucaine concentrations. Validation was performed across a range of CHE activities (1.5-22 kU/L) representing the three main genotypes, UU, UA, and AA. The respective DNs (mean +/- SD), determined on the Dimension-ES, were 82.0 +/- 1.6 (n = 32), 71.0 +/- 3.1 (n = 10), and 23.0 +/- 2.7 (n = 14), with corresponding imprecisions (CV) of 0.3%, 0.6%, and 5.2% (intraassay) and 0.7%, 0.7%, and 8.6% (interassay). Comparisons with reference (x) laboratory values and the DuPont aca (x') procedure (n = 53) gave regression equations of: y = 0.88x + 11.2, r = 0.99, and y = 0.85x' + 11.9, r = 0.99. A separate trial conducted with a Dimension-AR instrument gave similar performances. We conclude that the new DN method is fast, efficient, and appropriate for clinical use.