O-Ethylcholine (ethylcholine ether, EtCh), O-(2-chloroethyl)choline (ClEtCh), O-(2-bromoethyl)choline (BrEtCh), O(2-iodoethyl)choline (IEtCh), and O-(2-hydroxyethyl)choline (OHEtCh) were synthesized, and their toxicological and pharmacological activities were studied. The toxicities of these agents in male Swiss mice decrease in the following order: IEtCh > BrEtCh ⋍ ClEtCh > EtCh ⪢ OHEtCh. The animals died within 3 min of iv injection of the lethal dose, exhibiting a dramatic decrease in respiratory rate. Peripheral toxic signs, salivation, lacrimation, urination, and defecation, were abolished by atropine. Atropine partially protected mice against the lethality of EtCh as was evident from the increase in its LD50 by about 68% after atropine treatment. It completely inhibited respiratory depression caused by EtCh. Hexamethonium or d-tubocurarine did not alter the LD50 of these ethers. The respiratory depression caused by choline ethers other than EtCh at LD50 doses was significantly lowered by atropine pretreatment but was not abolished completely. The results suggest that these ethers produce the toxic effects by other mechanisms besides stimulating the muscarinic receptors, which are mainly involved in the maintenance of respiration. The effects of the choline ethers on the muscarinic and nicotinic receptors of the guinea pig longitudinal ileal muscle (GPI) were also studied to understand the relationship of the cholinergic system to the toxicity of these ethers. Their ED50s (μ m) to cause contraction of GPI in the presence of hexamethonium (37 μ m) have the following order (muscarinic activities): acetylcholine (ACh) (0.3) < EtCh (0.72) < ClEtCh ⋍ BrEtCh ⋍ IEtCh (7.8) ⪡ OHEtCh (56). Their relative maximal effects were higher than those of ACh (1.12 to 1.51). These compounds exhibited no significant inhibition of choline acetyltransferase and cholinesterase from GPI at the above ED50s. Hexamethonium increased the ED50s of the choline ethers by 1.4−2.2 times, indicating that they stimulate nicotinic receptors in Auerbach plexus. Atropine (>1 μM) blocked the contractions caused by all compounds. The steric volume occupied by the O-alkyl groups of the ethers is larger than the acetyl group of ACh, and the electron density around ether-oxygen is influenced by various substituents. Therefore, the charge and/or steric factors are limiting the muscarinic potencies of these choline ethers.