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Dual‐column headspace gas chromatographic analysis with two flame‐ionization detectors is a commonly used analytical technique for forensic blood ethanol quantitation. This technique is also applicable to the identification and quantitation of other volatile organic compounds such as methanol in biological samples. Compound identification by retention time is limited to those compounds with known retention times programmed into the instrument method. Historically, an early‐eluting peak from an unidentified compound has been observed in both chromatograms from antemortem blood samples analyzed for ethanol concentration with this technique. The unidentified compound's retention time matches that of methanol on one column but not on the second column. This previously unidentified compound has been identified as isobutylene. The proposed source of the isobutylene contamination historically observed in antemortem blood samples collected in 10‐ml gray‐top blood collection tubes is the conventional rubber stopper. Isobutylene was detected in deionized water stored in each of the seven lots of 10‐ml blood tubes tested; the expiration dates of the tubes tested spanned the years 2002–2022. Misidentification of isobutylene as methanol is possible when using a single‐column gas chromatographic system. The presence of isobutylene in blood collected in a gray‐top collection tube does not represent laboratory contamination, is not an interferent with blood ethanol quantitation, and does not affect the ethanol concentration in the blood. A 0.150 g/dl aqueous ethanol standard was stored in a gray‐top tube to evaluate the potential impact of isobutylene on ethanol quantitation. The solution's average ethanol concentration measured after storage was 0.150 g/dl. [ABSTRACT FROM AUTHOR]