The metabolic fate of 2,4-dichlorophenoxyacetic acid (2,4-D)-n-butyl ester in rats has not been extensively studied. Upon subcutaneous administration of a 100 mg/kg dose of 2,4-D butyl ester to four male Wistar rats, urine samples were analyzed by three analytical techniques for the presence of the butyl ester and metabolites. Thin layer chromatography (TLC), gas chromatography using an electron capture detector (GC-ECD), and gas chromatography-mass spectroscopy (GC-MS) were the techniques employed. 2,4-D butyl ester was rapidly hydrolyzed in the body to form 2,4-D acid. Ninety-five percent of the administered dose was excreted into the urine as the free acid within 48 h of injection, while only a small fraction (5%) was excreted over an additional 48 h. No amino acid conjugates or the parent 2,4-D butyl ester could be detected in the urine of treated rats. A minor metabolite (less than or equal to 2% of dose) was detected by GC-MS analysis of urine samples. This compound appears to be a side chain metabolite of the 2,4-D butyl ester. Some chemical properties of the metabolite were characterized, and a 2,4-D hydroxyethyl ester structure proposed. The mechanism of this minor metabolic formation remains unknown.