A rapid and precise colorimetric method for the determination of bovine red-blood-cell (RBC) cholinesterase (Ch E) activity, employing whole blood as the enzyme source, is presented. Whole blood determinations on 110 animals of mixed sexes of varied ages and breeds demonstrated a mean activity of 1.47 [plus or minus] 0.22 micromoles of acetylcholine (ACh) hydrolyzed by 50 micro-liters of whole blood, incubated for 10 minutes at 37[degree]C with a substrate concentration of 0.002 M acetyl choline chloride (total substrate 4 micro-moles). Under these conditions nearly 96% of the Ch E activity of whole blood is due to RBC Ch E. The effect of both the time and the blood volume (enzyme concentration) on hydrolysis of ACh was determined and a linear relationship was found to exist beyond the 50 microliters volume of whole blood and 10-minute time interval employed in this study. The method gives good agreement with the electrometric RBC method in measuring in vivo inhibition by organophosphorus insecticides.