This investigation examined the effectiveness of rehydration with glycerol and studied selected endocrine, cardiovascular and thermoregulatory responses during subsequent exercise in the heat. Eight highly-trained male cyclists completed three dehydration-rehydration experimental trials, that differed only in the rehydration treatment, each separated by seven days. Prior to each experimental day, subjects dehydrated to -4% of their body weight (bw), by exercise and water restriction. The experimental treatments were: no fluid (NF), glycerol (G) bolus (1g/kg bw) followed by water, and water alone (W). Rehydration (3% body weight) was given over an 80-min period. After rehydration, subjects cycled (74% VO_2peak) to exhaustion in a hot and wet (37°C & 48% rh) environment. For G, plasma volume was expanded (P<0.05) during rehydration and remained higher than W (P<0.05) during exercise. Exercise time to exhaustion during G (33±4 min) was longer (p<0.05) compared to both W (27±3 min) and NF (19±3 min). Cutaneous vascular conductance was significantly elevated (P<0.05) during G, but G provided no other thermoregulatory or cardiovascular benefits compared to W and NF. Fluid-regulating hormones (vasopressin, aldosterone, atriopeptin and plasma renin activity) decreased during rehydration and increased during exercise (except atriopeptin), but there were no differences between G and W. These data suggested that glycerol had little or no major effect on fluid regulating factors during rehydration or exercise, and the improved exercise capacity in G was likely due to a greater plasma volume during exercise.