Identifying metabolites associated with anaerobic hydrocarbon biodegradation is a reliable way to garner evidence for the intrinsic bioremediation of problem contaminants. While such metabolites have been detected at numerous sites, the in situ rates of anaerobic hydrocarbon decay remain largely unknown. Yet, realistic rate information is critical for predicting how long individual contaminants will persist and remain environmental threats. Here, single-well push–pull tests were conducted at two fuel-contaminated aquifers to determine the in situ biotransformation rates of a suite of hydrocarbons added as deuterated surrogates, including toluene-d8, o-xylene-d10, m-xylene-d10, ethylbenzene-d5 (or -d10), 1, 2, 4-trimethylbenzene-d12, 1, 3, 5-trimethylbenzene-d12, methylcyclohexane-d14 and n-hexane-d14. The formation of deuterated fumarate addition and downstream metabolites was quantified and found to be somewhat variable among wells in each aquifer, but generally within an order of magnitude. Deuterated metabolites formed in one aquifer at rates that ranged from 3 to 50 µg l−1 day−1, while the comparable rates at another aquifer were slower and ranged from 0.03 to 15 µg l−1 day−1. An important observation was that the deuterated hydrocarbon surrogates were metabolized in situ within hours or days at both sites, in contrast to many laboratory findings suggesting that long lag periods of weeks to months before the onset of anaerobic biodegradation are typical. It seems clear that highly reduced conditions are not detrimental to the intrinsic bioremediation of fuel-contaminated aquifers.