Naturally occurring biodegradation of petroleum hydrocarbons in the vadose zone depends on the physical soil environment influencing field-scale gas exchange and pore-scale microbial metabolism. In this study, we evaluated the effect of soil physical heterogeneity on biodegradation of petroleum vapors in a 16-m-deep, layered vadose zone. Soil slurry experiments (soil/water ratio 10:30 w/w, 25°C) on benzene biodegradation under aerobic and well-mixed conditions indicated that the biodegradation potential in different textured soil samples was related to soil type rather than depth, in the order: sandy loam > fine sand > limestone. Similarly, O2 consumption rates during in situ respiration tests performed at the site were higher in the sandy loam than in the fine sand, although the difference was less significant than in the slurries. Laboratory and field data generally agreed well and suggested a significant potential for aerobic biodegradation, even with nutrient-poor and deep subsurface conditions. In slurries of the sandy loam, the biodegradation potential declined with increasing in situ water saturation (i.e., decreasing air-filled porosity in the field). This showed a relation between antecedent undisturbed field conditions and the slurry biodegradation potential, and suggested air-filled porosity to be a key factor for the intrinsic biodegradation potential in the field.