A single dose (200 mg/kg body weight, i.p.) of 2,4-dichlorophenoxyacetic acid (2,4-D), commonly used as a herbicide, caused significant decreases in acetylcholinesterase (AChE) activity in diaphragm and other muscles of the rat. The 4S, 10S, and 16S forms of AChE were affected. The effect was maximal 15 to 24 h after injection. Choline acetyltransferase (CAT) activity was not affected. Neither AChE nor CAT activities changed in sciatic nerve from 2,4-D-treated animals. Spontaneous locomotor activity decreased dramatically 4 h after 2,4-D treatment. Myotonia that was present 1.5 h after 2,4-D injection became maximal at 2 to 6 h. Twenty-four hours after drug injection, when animals were recovering from myotonia, spontaneous locomotor activity was still depressed to 50% of control values. Prolonged distal motor latencies were observed 15 to 24 h after drug administration. AChE activity and spontaneous locomotor activity returned to control values at 48 h. Thus, 2,4-D causes a decrement of end-plate AChE, as well as behavioral and electrophysiologic changes. Decreased activity of AChE may be an early step in development of the myopathy that occurs after large dose 2,4-D.