The activity of tertiary and quaternary anticholinergic drugs was compared in two different test procedures designed to measure cholinolytic activity in mice. The four drugs utilized were atropine sulfate, atropine methylnitrate, scopolamine hydrobromide, and scopolamine methylnitrate. The results led to the conclusion that one of these test procedures, the induction of mydriasis (increase in pupil size), primarily measures peripheral anticholinergic activity whereas the other procedure, inhibition of phy‐sostigmine lethality, primarily measures anticholinergic activity in the CNS. These two test procedures can be utilized to characterize the nature of the cholinolytic properties of prospective therapeutic drug candidates.