3-O-methyl-D-glucose has been extensively used as a proxy for d-glucose uptake. This nonmetabolizable analog has lower affinity for transporters, potentially leading to underestimates of glucose absorption rates as well as overestimates of the nutritional significance of passive uptake. Here we sought to precisely quantify the bias, if any, incurred when using 3-O-methyl-D-glucose by comparing relative absorption rates with D-glucose in vivo in a seasonally frugivorous bird, the American robin. By simultaneously administering these D-glucose probes with L-glucose--the latter absorbed only via nonmediated mechanisms and the former absorbed by both mediated and nonmediated mechanisms--using common pharmacokinetic procedures, we were able to estimate the nutritional significance of paracellular uptake in this species. The relative absorption rate of 3-O-methyl-D-glucose calculated over the initial absorptive phase was not significantly different from that of D-glucose, indicating that the former provides reasonable estimates of glucose absorption rates in vivo. The ratio of L-glucose to D-glucose cumulative fractional absorption indicates that around 60% of total glucose absorption in American robins is paracellular and showed no apparent bias in using 3-O-methyl-D-glucose when averaged over the entire initial absorptive phase. Although the absorption and elimination kinetics of radiolabeled D-glucose were appropriate for pharmacokinetic analysis in this study, because of the potential for interspecific differences in loss to catabolism, it should be used in vivo with caution and with independent verification of absorption efficiency.