Daily treatment of rats with 2,4-dithiobiuret (DTB, 1 mg/kg) produces an apparent flaccid muscle weakness that begins in the hindlimbs and ascends to involve muscles of the trunk, forelimbs, head, and neck. The insidious onset and progressive nature of the apparent weakness were detected by impaired rotarod performance, loss of righting response, and death after median cumulative doses of 4.9, 7.1, and 8.0 mg/kg, respectively, in male rats. If DTB treatment was stopped after the first day of rotarod failure, male rats recovered from impaired rotarod performance in 2–5 days. Sensitivity to DTB-induced rotarod failure was greatest in older rats of both sexes. Females were more susceptible than males to DTB-induced rotarod failure and frequently died after treatment was stopped, whereas males recovered. Effects of chronic DTB treatment that accompanied the impairment in rotarod performance, but did not appear to cause it, were decreased food and water intake, loss of body weight, diuresis, chromodacryorrhea, and watery, mucoid feces. Whole blood cholinesterase activity was not significantly altered and tail flick, corneal, vibrissae, pinna, and foot with-drawal responses could be elicited in animals that failed the rotarod test. The LD 50 determined 48 hr after a single dose of DTB was 29 mg/kg yet acute treatment with up to 20 mg/kg did not affect rotarod performance when rats were tested for up to 3 weeks after treatment. The site and mechanism by which DTB impairs rotarod performance in the rat is unknown.