To assess the relative contributions of gluconeogenesis and glycogenolysis to overall hepatic glucose output in postabsorptive normal humans and those of the indirect and direct pathways for glycogen synthesis, we studied six normal volunteers, who had been fasted for 16 h to reduce their hepatic glycogen stores, and then ingested glucose (250 g over 10 h) enriched with [6-3H] glucose to replenish and label their hepatic glycogen. After a 10-h overnight fast, release of the [6-3H] glucose into the circulation was traced with [2-3H] glucose to estimate breakdown of glycogen that had been formed via the direct pathway while gluconeogenesis was simultaneously estimated by incorporation of infused [14C] lactate into plasma glucose. We found that release of [6-3H] glucose into plasma (6.79 +/- 0.69 mumol.kg-1.min-1) accounted for 46 +/- 5% of hepatic glucose output (15.0 +/- 0.7 mumol.kg-1.min-1) while glucose formed from lactate (2.71 +/- 0.28 mumol.kg-1.min-1) accounted for 19 +/- 2% of hepatic glucose output. Since these determinations underestimate direct pathway glycogenolysis and overall gluconeogenesis, a maximal estimate for the contribution of indirect pathway glycogenolysis to hepatic glucose output is obtained by subtracting the sum of direct pathway glycogenolysis and lactate gluconeogenesis from hepatic glucose output. This amounted to a maximum of 36 +/- 5% of hepatic glucose output and 44 +/- 6% of overall glycogenolysis.(ABSTRACT TRUNCATED AT 250 WORDS)