The herbicide 2,4-Dichlorophenoxyacetic acid (2,4-D) is a commonly used herbicide and one component of Agent Orange. The herbicide is commonly formulated as the butyl ester. The effects of a 50:50 mixture of n-butyl and iso-butyl esters of 2,4-D (2,4-D mixed butyl esters, 150-175 mg/kg/day SC) on photocell locomotor activity and landing foot splay were assessed in rats. Similarly the effects of 2,4-D mixed butyl esters and pure 2,4-D-n-butyl ester (150 mg/kg/day SC) on photocell locomotor activity were assessed in both food deprived and free feeding rats. In general, food deprivation tended to decrease the sensitivity of rats to the effects of either formulation. The spectrum of neurobehavioral effects varied with the ester isomers. Both 2,4-D-n-butyl ester and 2,4-D mixed butyl esters depressed photocell locomotor activity. 2,4-D mixed butyl esters failed to increase landing foot splay as reported for 2,4-D-n-butyl ester. The extent of ester hydrolysis was similar when comparable concentrations of 2,4-D acid were measured in blood and brain four hours following either formulation. 2,4-D-n-butyl ester caused significantly more activity depression than 2,4-D mixed butyl esters. Additionally, tolerance developed more rapidly for animals receiving 2,4-D mixed butyl esters than for animals receiving 2,4-D-n-butyl ester. These studies exemplify the importance of herbicide formulation and subject nutritional status in the expression of neurobehavioral toxicity.