Histological evidence of tetracycline use has been reported in an ancient X-Group population (350–550 CE) from Sudanese Nubia (Bassett et al., 1980). When bone samples were examined by fluorescent microscopy under UV light at 490 Å yellow–green fluorophore deposition bands, similar to those produced by tetracycline, were observed, suggesting significant exposure of the population to the antibiotic. These reports were met skeptically with claims that the fluorescence was the result of postmortem taphonomic infiltration of bacteria and fungi. Herein, we report the acid extraction and mass spectroscopic characterization of the antibiotic tetracycline from these samples. The bone samples were demineralized in anhydrous hydrogen fluoride which dissolved the bone-complexed tetracycline, followed by isolation by solid phase extraction on reverse-phase media. Chemical characterization by high pressure liquid chromatography mass-spectroscopic procedures showed that the retention times and mass spectra of the bone extract were identical to tetracycline when treated similarly. These results indicate that a natural product tetracycline was detectable within the sampled bone and was converted to the acid-stable form, anhydrotetracycline, with a mass + H of 427.1 amu. Our findings show that the bone sampled is labeled by the antibiotic tetracycline, and that the NAX population ingested and were exposed to tetracycline-containing materials in their dietary regime. Am J Phys Anthropol 143:151–154, 2010. © 2010 Wiley-Liss, Inc.