Glucose plays a fundamental role in mammalian energetics but its contribution as a metabolic fuel is not well established for fish; the accurate in vivo measurement of glucose flux is essential to determine the importance of this substrate in the energy budget of teleosts. Therefore, the goal of the present study was to verify the reliability of the continuous tracer infusion method for estimating glucose turnover rate in rainbow trout Oncorhynchus mykiss. Our secondary goals were to determine whether glucose flux can be estimated more accurately from plasma or from whole-blood samples, and to obtain an estimate of renal glucose production. Continuous infusions of [6-3H]glucose were performed in hepatectomized and intact animals. In some hepatectomized individuals, liver glucose production was replaced by a pump infusing unlabelled glucose at a known rate. Renal glucose production was measured in hepatectomized fish where liver glucose production was not replaced, and it averaged 1.1±0.1µmolkg-1min-1 (mean ± s.e.m., N=5). Results show that glucose turnover rate is quantified accurately by continuous tracer infusion and that glucose flux can be estimated equally well from plasma (error of -0.7±4.9%) and from whole-blood (error of -5.7±2.9%) samples (means ± s.e.m., N=7). This study provides the first experimental validation of continuous tracer infusion in fish, and shows that this method could become a powerful tool to investigate hormonal regulation of glucose metabolism in live teleosts.