This study tests the hypothesis that the metabolic and endocrine shift characterizing the phase II-phase III transition during prolonged fasting is related to a decrease in fatty acid oxidation. Changes in the plasma concentrations of various metabolites and hormones, and in lipolytic fluxes as determined by continuous infusion of 2-[³H]glycerol and 1-[¹⁴C]palmitate, were examined in vivo in spontaneously fasting king penguins in the phase II status (large fat stores, protein sparing) before, during and after treatment with mercaptoacetate (MA), an inhibitor of fatty acid oxidation. MA induced a 7-fold decrease in plasma ß-hydroxybutyrate and a 2-2.5-fold increase in plasma non-esterified fatty acids (NEFA), glycerol and triacylglycerols. MA also stimulated lipolytic fluxes, increasing the rate of appearance of NEFA and glycerol by 60-90%. This stimulation might be partly mediated by a doubling of circulating glucagon, plasma insulin remaining unchanged. The plasma glucose level was unaffected by MA treatment. Plasma uric acid increased by 4-fold, indicating a marked acceleration of body protein breakdown, possibly mediated by a 2.5-fold increase in circulating corticosterone. The strong similarities between these changes and those observed at the phase II-phase III transition in fasting penguins support the view that entrance into phase III, and especially the end of protein sparing, is related to a decrease in fatty acid oxidation rather than to a reduced NEFA availability. MA could be therefore a useful tool for understanding the mechanisms underlying the phase II-phase III transition in spontaneously fasting birds and the associated stimulation of feeding behavior.