Oxotremorine sesquioxalate (2.0 × 10-5M), arecoline-HBr (2.0 × 10-5M), aceclidine-HCl (1 × 10-4M), and carbachol chloride (2.0 × 10-7M) potentiated the response of isolated frog rectus abdominis muscle to acetylcholine (I). Atropine sulfate (1.0 × 10-5M) inhibited the response of the preparation to I, and 1,4-bis-(hexamethyleneimino)-2-butyne-2HCl, a central anticholinergic agent, produced no significant change. The potentiation observed with muscarinic agents was more marked when the assay was conducted in the presence of frog brain extracts which had been boiled with alkali to destroy intrinsic I; these extracts also caused significant potentiation even in the absence of added muscarinic agents. ClO4- (>2 × 10-4M) also potentiated the response of the muscle preparation to I, but little potentiation was observed following the addition of K+. The greater potentiation observed with muscarinic agents in the presence of brain extracts may be due to an added effect between the pharmacol. agents and ClO4- (used in extraction of brain tissue). The use of HClO4 should thus be avoided as an extracting agent prior to bioassay of I due to a variable sensitization it produces, particularly when other drugs may be present. 16 references.