Estimation of glucose uptake in vivo using 2-deoxy-D-[2,6-3H]glucose (2DG) relies upon the assumption that the phosphorylated form, 2-deoxy-D-2,6-3H]glucose 6-phosphate (2DGP), cannot be further metabolised. We aimed to determine whether this assumption leads to underestimation of glucose uptake due to the incorporation of 2DGP into glycogen. Rats were infused with [U-14C]glucose and 2-[3H]DG, and the incorporation into glycogen was measured. These were compared to the accumulation of 2-[3H]DGP in heart, liver, muscle, white adipose tissue and brown adipose tissue. 2DG was incorporated into glycogen in an insulin-dependent manner (e.g. in soleus, at basal, physiological and supraphysiological insulin concentrations, glycogen synthesis rates from 2DG were 17.81 +/- 3.07, 64.47 +/- 7.47 and 203.23 +/- 44.52 nmol glycogen incorporated/g min-1, respectively). The rate of glycogen synthesis from 2-[3H]DG was identical to that for [U-14C]glucose in all tissues studied except for heart and brown adipose tissue (e.g. in soleus at physiological insulin concentration, 2-[3H]DG incorporation was 64.47 +/- 7.47 and [U-14C]glucose incorporation was 61.87 +/- 7.56 nmol glucose/g min-1). Furthermore, the proportion of 2DG incorporated into glycogen was significant with respect to total glucose uptake at all plasma insulin concentrations (10.7% +/- 0.9, 14.0 +/- 1.9 and 25.6% +/- 5.6 at basal, physiological and supraphysiological insulin concentrations, respectively). 2DG was metabolised to glycogen in all tissues studied causing an underestimation of the rate of glucose uptake by measurement of 2DGP accumulation alone. In addition, use of 2DG could provide a method for assessing the rate of direct glycogen synthesis in the rat.