The contribution of gluconeogenic precursors to renal glucose production (RGP) during insulin-induced hypoglycemia was assessed in conscious dogs. Ten days after surgical placement of sampling catheters in the right and left renal veins and femoral artery and an infusion catheter in the left renal artery, systemic and renal glucose and glycerol kinetics were measured with peripheral infusions of [6-3H]glucose and [2-13C]glycerol. Renal blood flow was determined with a flowprobe, and the renal balance of lactate, alanine, and glycerol was calculated by arteriovenous difference. After baseline, six dogs received 2-h simultaneous infusions of peripheral insulin (4 mU x kg(-1) x min(-1)) and left intrarenal [6,6-2H]dextrose (14 micromol x kg(-1) x min(-1)) to achieve and maintain left renal normoglycemia during systemic hypoglycemia. Arterial glucose decreased from 5.3 +/- 0.1 to 2.2 +/- 0.1 mmol/l; insulin increased from 46 +/- 5 to 1,050 +/- 50 pmol/l; epinephrine, from 130 +/- 8 to 1,825 +/- 50 pg/ml; norepinephrine, from 129 +/- 6 to 387 +/- 15 pg/ml; and glucagon, from 52 +/- 2 to 156 +/- 12 pg/ml (all P < 0.01). RGP increased from 1.7 +/- 0.4 to 3.0 +/- 0.5 (left) and from 0.6 +/- 0.2 to 3.2 +/- 0.2 (right) micromol x kg(-1) x min(-1) (P < 0.01). Whole-body glycerol appearance increased from 6.0 +/- 0.5 to 7.7 +/- 0.7 micromol x kg(-1) x min(-1)(P < 0.01); renal conversion of glycerol to glucose increased from 0.13 +/- 0.04 to 0.30 +/- 0.10 (left) and from 0.11 +/- 0.03 to 0.25 +/- 0.05 (right) micromol x kg(-1) x min(-1), (P < 0.05). Net renal gluconeogenic precursor uptake increased from 1.5 +/- 0.4 to 5.0 +/- 0.4 (left) and from 0.9 +/- 0.2 to 3.8 +/- 0.4 (right) micromol x kg(-1) x min(-1) (P < 0.01). Renal lactate uptake could account for approximately 40% of postabsorptive RGP and for 60% of RGP during hypoglycemia. These results indicate that gluconeogenic precursor extraction by the kidney, particularly lactate, is stimulated by counterregulatory hormones and accounts for a significant fraction of the enhanced gluconeogenesis induced by hypoglycemia.