The contribution of glycerol to glucose production has been measured in healthy volunteers by the simultaneous primed constant infusion of 1-[13C]glycerol and 3-[3H]glucose and the determination of the rates of appearance (Ra) of glycerol, glucose, and glycerol-derived glucose. In the postabsorptive state, glycerol Ra was 3.11 +/- 0.44 mumol.kg-1.min-1, of which 36% was converted to glucose, accounting for 4.5% of total glucose production. After 62-86 h of starvation, glycerol Ra rose to 5.32 +/- 0.58 mumol.kg-1.min-1, and 68% of glycerol was converted to glucose. This accounted for 21.6% of total glucose production. Glycerol Ra was closely correlated with its conversion and contribution to glucose. These findings confirm that the contribution of glycerol to glucose production is directly correlated to its release as a consequence of lipolysis and support the notion that the central physiological role of accelerated lipolysis in fasting is the provision of gluconeogenic precursor.