BACKGROUND: Oral glucosamine preparations are widely used as a treatment for osteoarthritis, purportedly functioning by a variety of mechanisms suggested by results of in vitro experiments, and generally using glucosamine concentrations well in excess of 100 micromol/l. OBJECTIVE: To use high performance liquid chromatography with a high sensitivity Metrohm-Peak instrument for pulsed amperometric measurement of human serum glucosamine; a detection limit of 0.5 micromol/l at 1:10 serum dilution allowed measurement of low levels of glucosamine in human serum, which previously has not been possible. METHODS: Eighteen subjects with osteoarthritis were given 1,500 mg of commercial glucosamine sulphate after an overnight fast, and serum was then obtained at baseline and every 15-30 minutes over 3 hours, and additionally, from two subjects at 5 and 8 hours. Urine samples were collected at baseline and 3 hours after ingestion from three subjects. RESULTS: Baseline glucosamine was below the detection limit of 0.5 mumol/l for all subjects, but after ingestion, glucosamine was detected in 17/18 subjects, beginning to rise at 30-45 minutes to a maximum at 90-180 minutes, with a range of 1.9-11.5 micromol/l (0.34-2 microg/ml). CONCLUSION: This maximum concentration of 11.5 micromol/l has previously been shown to contribute less than 2% of the galactosamine incorporated into chondroitin sulphate in incubations of glucosamine with cultured human chondrocytes, and is a much lower concentration than the glucosamine concentrations claimed by other investigators to have various significant in vitro effects. This raises questions about current biological rationales for glucosamine use that were based on in vitro effects of glucosamine at much higher concentrations.