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Acetoacetate concentration in plasma was determined by head-space gas chromatography after decarboxylation by the use of acetoacetate decarboxylase, which was recently isolated from Bacillus polymyxa A-57 strain. Partial purification of acetoacetate decarboxylase solution was carried out by DEAE-cellulose column chromatography of the cell-free extract, which was prepared by ultrasonication. The properties of the enzyme were found to be particularly suitable for acetoacetate assay (optimum pH, 5.8 ; Vmax, 230μmol/min/mg ; Km, 5.9×10-4M). Plasma was deproteinized by Somogyi's method. Acetone in the mildly basic supernatant was assayed by head-space gas chromatography. Acetoacetate was calculated by subtracting blank acetone from total acetone after enzymatic decarboxylation of acetoacetate. The minimal detectable concentration was 1μM. This method gave better reproducibility (CV=2.0-8.0%) and recovery (96.0-101.8%) than chemical decarboxylation with perchloric acid. Normal subjects (n=31) showed plasma acetone levels of 7.2±3.4μM and acetoacetate levels of 22.5±9.7μM. Diabetic patients (n=44), treated by diet control alone without drug therapy, gave plasma acetone levels of 8.1±3.5μM and acetoacetate levels of 25.0±8.0μM. There was no significant difference between the two groups.