Neonatal and adult rat islets, cultured for 7–9 days in the presence of 10.5 mM d-glucose, were incubated for 120 min with either d-glucose (2.8 and 16.7 mM) or l-leucine (1.0 and 20.0 mM). The total and anaerobic rates of glycolysis, as judged respectively through the generation of ³H₂O from d-[5-³H]glucose and ¹⁴C-labelled lactate from d-[3,4-¹⁴C]glucose or d-[6-¹⁴C]glucose were higher in neonatal than adult islets, but increased to a lesser relative extent in neonatal than adult islets in response to a rise in hexose concentration. The flow through the pentose phosphate pathway, as judged from the difference between d-[1-¹⁴C]glucose and d-[6-¹⁴C]glucose oxidation was higher in neonatal than adult islets. The flow through the reaction catalyzed by pyruvate dehydrogenase, as judged from the oxidation of d-[3,4-¹⁴C]glucose, was lower in neonatal than adult islets incubated in the presence of 16.7 mM (but not 2.8 mM) d-glucose. The oxidation of acetyl residues relative to their generation rate, as judged from the ratio of d-[6-¹⁴C]glucose to d-[3,4-¹⁴C]glucose oxidation, was not affected by the hexose concentration whether in neonatal or adult islets, but was about twice higher in the latter than former islets. The rate of d-[6-¹⁴C]glucose oxidation was also higher in adult than neonatal islets, especially at the high concentration of d-glucose. In both neonatal and adult islets, a rise in hexose concentration stimulated preferentially the oxidation of d-[3,4-¹⁴C]glucose or d-[6-¹⁴C]glucose relative to the utilization of d-[5-³H]glucose. Inversely, the absence of Ca²⁺ and presence or cycloheximide inhibited preferentially d-[6-¹⁴C]glucose oxidation relative to d-[5-³H]glucose utilization. Such a preferential inhibition was observed both at low and high concentrations of d-glucose in neonatal islets, but only at the high hexose concentration in adult islets. The generation of ¹⁴C-labelled acidic metabolites and ¹⁴CO₂ from l-[U-¹⁴C]leucine was higher in neonatal than adult islets exposed to a low concentration of the amino acid. However, a rise in l-leucine concentration increased its catabolism to a lesser extent in neonatal than adult islets. The absence of Ca²⁺ and presence of cycloheximide again caused a preferential inhibition of l-[U-¹⁴C]leucine oxidation relative to its conversion to acidic metabolites in neonatal islets incubated at low or high concentration of l-leucine, but only exerted a comparable effect in adult islets exposed to a high concentration of the amino acid. These findings suggest that neonatal islets are actively engaged into energy-consuming anabolic processes even when exposed to low concentrations of d-glucose and l-leucine. However, in response to a rise in either d-glucose or l-leucine concentration, the generation of ATP is increased to a lesser extent in neonatal than adult islets, and this may in turn account for a lesser secretory response to these nutrient secretagogues.