Although a large amount of soil carbon (C) is stored in subsurface soils, most studies on soil C dynamics focus on the upper layers. The aim of this study is to assess the factors that regulate C mineralization in mountain grassland soils under standard laboratory conditions to compare regulation mechanisms at surface and subsurface horizons. For this purpose soil samples of surface and subsurface horizons from 35 locations were incubated under laboratory conditions, CO2 efflux rates were measured and microbial biomass C (MBC) and net N mineralization were determined. We also analysed the samples for pH, extractable C after fumigation (Cfe), potentially mineralizable N (PMN), reactive and non-reactive P, sum of exchangeable bases and clay content in order to assess the influence of soil characteristics on C mineralization. The influence of climate of each site on soil C mineralization under the same laboratory conditions was also explored for surface and subsurface horizons. C mineralization in surface horizons related positively with Cfe content, suggesting that microbial activity in this horizon was mainly regulated by the availability of C. By contrast, in subsurface horizons, C mineralization related with PMN and was independent of measured C fractions, suggesting that microbial activity in subsurface horizons was limited by the availability of N and that the available forms of C were more stable in these horizons. The effects of local climate on laboratory C mineralization were significant in both soil horizons, with lower rates of C mineralization being recorded in soils from wetter and warmer sites. This fact, suggested that the C stabilisation mechanisms in mountain grassland soils may be affected by the climate in which soils develop.