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The response of soil CO2 fluxes (Rsoil) 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 Rsoil through changes to Michaelis-Menten parameters (i.e. Vmax and Km). Results of this study demonstrate that Rsoil response to N enrichment and changes in temperature are dependent on the C availability of soil substrates. N addition influenced Rsoil through both the maximum rate (Vmax) and the half saturation constant (Km). The increase in Km corresponded to a decrease in Rsoil when C was limited. Alternatively, when C was abundant, N enrichment increased Rsoil, which corresponded to an increase in Vmax. Regulation of temperature sensitivity through Vmax and Km was also dependent on C availability. Both Vmax and Km 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 Rsoil and justifying the use of Michaelis-Menten kinetics in biogeochemical modeling [ABSTRACT FROM AUTHOR]