Abstract: The cited determination was carried out with use of a flow injection system incorporating a robotic sample preparation and injection unit, a spectrophotometer and an Apple computer. Plasma or haemolysate (5 µL) was mixed with 0.1 M phosphate buffer (pH 7.5) and 10 µL of substrate solution (succinylcholine, butyrylcholine or acetylcholine) and the mixture was heated at 30°C for 20 min. The reaction was stopped by the addition of 10 µL of physostigmine. The choline formed was determined by addition of a color reagent, containing choline oxidase (forming H2O2), peroxidase, phenol and 4-aminoantipyrine and, after further heating, the absorbance was measured at 500 nm. The calibration graph was rectilinear for 0.5 to 4 mM choline. The results using the three substrates correlated well (r 0.94), and the between-day coefficient of variation for plasma analysis was 7% (n = 12). The preferred substrate is succinylcholine. The use of the flow injection system decreases the volume of reagent and sample solution required by a factor of ten.