The response of soil CO2 fluxes (R-soil) to interactions between carbon (C) and nitrogen (N) availability or C and temperature conditions is not well understood, but may increasingly affect future C storage under the combined anthropogenic impacts of N deposition and climate change. Here we addressed this uncertainty through a series of laboratory incubation experiments using soils from three contrasting ecosystems to investigate how changes in C, N, and temperature regulate R-soil through changes to Michaelis-Menten parameters (i.e. V-max and K-m). Results of this study demonstrate that R-soil response to N enrichment and changes in temperature are dependent on the C availability of soil substrates. N addition influenced R-soil through both the maximum rate (V-max) and the half saturation constant (K-m). The increase in K-m corresponded to a decrease in R-soil when C was limited. Alternatively, when C was abundant N enrichment increased R-soil, which corresponded to an increase in V-max. Regulation of temperature sensitivity through V-max and K-m was also dependent on C availability. Both V-max and K-m demonstrated positive temperature responses, supporting the hypothesis of a canceling effect at low C concentrations. While temperature sensitivity was influenced by both C quantity and C complexity, our results suggested that C quantity is a stronger predictor. Despite strong differences in climate, vegetation, and management of our soils, C-N and C-temperature interactions were markedly similar between sites, highlighting the importance of C availability in the regulation of R-soil and justifying the use of Michaelis-Menten kinetics in biogeochemical modeling. (C) 2015 Elsevier Ltd. All rights reserved.