The isotopic signatures of crude oil hydrocarbons are potentially powerful intrinsic tracers to their origins and the processes by which the oils are modified in the environment. Stable carbon isotopic data are of limited use for studying petroleum contaminants because of the relatively small amount of isotopic fractionation that occurs during natural processes. Hydrogen isotopes, in contrast, are commonly fractionated to a much greater extent and as a result display larger variations in delta values. We studied the effect of in vitro aerobic biodegradation on the hydrogen isotopic composition of individual n-alkanes from crude oil. The isotopic analysis was conducted using gas chromatography-thermal conversion-isotope ratio mass spectrometry. In general, biodegradation rates decreased with increasing hydrocarbon chain length, consistent with previous studies. More importantly the n-alkanes that were degraded at the fastest rates (n-C15 to n-C18) also showed the largest overall isotopic fractionation (approximately 12-25 per thousand deuterium enrichment), suggesting that the lower molecular weight n-alkanes can be used to monitor in-situ bioremediation of crude oil contamination. The hydrogen isotopic compositions of the longer chain alkanes (n-C19 to n-C27) were relatively stable during biodegradation (<5%o overall deuterium enrichment), indicating that these compounds are effective tracers for oil-source identification studies.