In order to avoid the use of radioactive tracers for the determination of human ketone body turnover, we have developed a method using a primed-continuous infusion of 13C-labelled acetoacetate or D-beta-hydroxybutyrate. Determination of the mole percent enrichment of blood acetoacetate and D-beta-hydroxybutyrate was performed by gas chromatography/mass spectrometry. In the post-absorptive state, the mean total ketone body appearance rate, determined in four subjects, was 3.74 mumol X kg-1 X min-1 using [3,4-13C2] acetoacetate and 2.76 mumol X kg-1 X min-1 using [3-13C]D-beta-hydroxybutyrate, values in agreement with those reported in studies with 14C-labelled tracers. In order to evaluate the usefulness of the method for determination of ketone body kinetics in non steady-state conditions, we infused four subjects with natural sodium acetoacetate and calculated the isotopically determined total ketone body appearance rate using a single compartment model (volume of distribution 0.20 l/kg; functional pool fraction: 1). During the tests with [3,4-13C2]-acetoacetate, the actual infusion rates of natural acetoacetate were 7.3 +/- 0.3, 14.6 +/- 0.8, 21.9 +/- 1.2 and 10.9 +/- 0.6 mumol X kg-1 X min-1 whereas the corresponding isotopically determined total ketone body appearance rates were respectively 9.2 +/- 1.0, 16.3 +/- 0.7, 23.1 +/- 1.1 and 10.7 +/- 0.8 mumol X kg-1 X min-1. During the tests with [3-13C]D-beta-hydroxybutyrate, the actual infusion rates were 8.4 +/- 0.5, 16.8 +/- 0.9, 25.2 +/- 1.4 and 12.6 +/- 0.8 mumol X kg-1 X min-1, and the isotopically determined appearance rates respectively 11.1 +/- 0.7, 16.7 +/- 0.7, 25.0 +/- 1.1 and 11.1 +/- 0.7 mumol X kg-1 X min-1.(ABSTRACT TRUNCATED AT 250 WORDS)