It is estimated that in excess of 50% of the soil carbon stock is found in the subsoil (below 20–30 cm). Despite this very few studies have paid attention to the subsoil. Although surface and subsurface horizons differ in pedological, environmental and physicochemical features, which are all likely to affect the mechanisms and biological actors involved, models of carbon dynamics tend to assume that the underlying processes are identical in all horizons, but with lower gross fluxes in the subsurface. The aim of this study was to test this assumption by analysing factors governing organic matter decomposition in topsoil (from depths of 5–10 cm) and subsoil (from depths of 80–100 cm). To this end, we established incubations that lasted 51 days, in which factors that were thought to control organic matter mineralization were altered: oxygen concentration, soil structure and the energetic and nutritional status. At the end of the incubation period, the microbial biomass was measured and the community level physiological profiles established. The mineralization per unit organic carbon proved to be as important in the subsoil as it was in surface samples, in spite of lower carbon contents and different catabolic profiles. Differences in the treatment effects indicated that the controls on C dynamics were different in topsoil and subsoil: disrupting the structure of the subsoil caused a 75% increase in mineralization while the surface samples remained unaffected. On the other hand, a significant priming affect was found in the topsoil but not in the subsoil samples. Spatial heterogeneity in carbon content, respiration and microbial communities was greater in subsoil than in topsoil at the field scale. These data suggest greater attention should be paid to the subsoil if global C dynamics is to be fully understood.