The objective of this study was to determine the urinary excretion of methylxanthines in horses following ingestion of chocolate over eight days. The study was performed in response to gas chromatography-mass spectrometry (GC-MS) confirmation of the presence of caffeine in a positive urine test in a racehorse. The trainer of the horse alleged that he often administered chocolate-coated peanuts as treats to his horses, and he believed that the ingestion of chocolate was responsible for the positive urine test. The urinary excretion of theobromine and caffeine after the ingestion of chocolate-coated peanuts was investigated in three horses. Enzyme-linked immunoassay (ELISA), high-performance liquid chromatography (HPLC), and GC-MS assays were performed on all urine specimens. Theobromine (HPLC) was detected for 72 h and caffeine (GC-MS) for 48 h after chronic ingestion of chocolate-coated peanuts. Methylxanthines were detected by ELISA for 120 h after administration of chocolate.