The effects of fasting for 24 h and 48 h on D-3-hydroxybutyrate utilization and acetoacetate, L-lactate and pyruvate production by the isolated non-working perfused rat heart were investigated over a wide range of DL-3-HB concentrations. D-3-HB utilization is concentration dependent and shows saturation kinetics, D-3-HB oxidation is correlated with D-3-HB concentration. Acetoacetate production is proportional to DL-3-HB concentration. L-lactate production is proportional to DL-3-HB concentration up to 5 mM following a 24 h fast and up to 10 mM after a 48 h fast, but further increase in DL-3-HB concentration decreases the rate of lactate production. Fasting enhances D-3-HB utilization at 16 mM DL-3-HB by 16% and 25% in 24 h and 48 h fast respectively, but has no significant effect at lower concentration. Fasting has no effect on acetoacetate production. Fasting for 48 h doubled the half-saturation concentration (Ku) without significant change in the maximum rate of utilization (Vu) of D-3-HB.