We describe here a new method for the analysis of alkanes ( n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-undecane, and n-dodecane) in blood using headspace solid-phase microextraction gas chromatography/mass spectrometry. This method is used to measure picogram per milliliter levels of n-alkanes in blood that may result from nonoccupational exposure to alkanes and other volatile nonpolar compounds from common sources such as petroleum-based fuel. This alkane signature is useful in distinguishing typical fuel biomarkers (e.g., benzene and toluene) from other confounding exposure sources such as cigarette smoke. Development of this method required special attention to sample handling as alkanes are not highly soluble in aqueous matrixes and exist as ubiquitous compounds found in many laboratory materials and the environment. In particular, significant n-hexane contamination ( approximately 0.4 ng/mL) occurred from collecting blood samples in vacutainers. This residue was removed by boiling the vacutainer stoppers in methanol followed by vacuum baking. For all the alkanes, the calculated accuracy demonstrated for the water-based standards ranged from 3.3% to 17% as deduced from the difference of the lowest and middle standards from the curve fit. Quality control data among runs over a 10 month period were found to vary from 14% to -29%, with a few exceptions. The resulting quantification limits for n-hexane through n-decane ranged from 0.069 to 0.132 ng/mL. In the analysis of 1200 blood samples from people with no known occupational exposure, median blood levels for all n-alkanes were below these quantification limits. n-Hexane levels above the method detection limit were, however, found in 1.3% of the samples.