During our extensive surveillance of opiates in urine specimens of opium users, we noticed the appearance of an unknown peak (compound X) in total ion chromatograms obtained by gas chromatography-mass spectrometry (GC-MS) after enzymatic hydrolysis and trimethylsilyl (TMS) derivatization. We identified the compound X as oripavine. Oripavine was found to be a new and useful putative marker of opium/poppy seed use in differentiation from heroin, pharmaceutical codeine, and pharmaceutical morphine use. The presence of oripavine in the urine of opium users is probably the result of O-demethylation of the opium alkaloid thebaine. Analytical method optimization for GC-MS detection of oripavine in urine was also undertaken. Underivatized oripavine could not be detected by GC-MS, and trials for derivatization of oripavine with acetic anhydride and propionic anhydride were unsuccessful. Trials were successful with bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane. It was also disclosed that almost all amounts of oripavine in human urine existed in the unconjugated form; it was absolutely necessary to hydrolyze the conjugate before TMS derivatization of oripavine for its GC-MS analysis. © 2011 Japanese Association of Forensic Toxicology and Springer.