Headspace solid-phase microextraction (HS-SPME) combined with gas chromatography/pyrolysis-gas chromatography/combustion-isotope ratio mass spectrometry (GC/Py-GC/C-IRMS) was developed for the simultaneous determination of the intramolecular and molecular carbon-isotopic composition (δ(13) C value) of acetic acid. The δ(13) C values of carboxyl and methyl carbon were standardized using calibration curves constructed from the regression between the measured δ(13) C values and the δ(13) C values of working standards determined in a previous study. We applied this developed HS-SPME-GC/Py-GC/C-IRMS technique to commercial vinegars. In one injection analysis, the bulk and intramolecular δ(13) C values of pure acetic acid standards can be obtained. The repeatability (1σ) of the bulk δ(13) C values is within ±0.4‰, and that of the δ(13) Ccarboxyl and δ(13) Cmethyl values is within ±0.6‰. The intramolecular δ(13) C values of acetic acid in vinegars exhibit a similar pattern. The average Δδ value (δ(13) CCOOH - δ(13) CCH3 ) is 4.3 ± 2.0‰. The approach presented herein for the molecular and intramolecular δ(13) C determination of acetic acid avoids switching between configuration systems and thereby reduces systematic errors. It is expected to be useful for examining isotope fractionation associated with processes related to organic acid (bio)transformations.