Adoption of less invasive management practices, such as no-till (NT) and continuous cropping, could reduce CO2 emissions from agricultural soils by retaining soil organic matter (SOM). We hypothesized that C storage increases as cropping intensity increases and tillage decreases. We also hypothesized that pulsed addition of C increases the mineralization of native SOM. We evaluated C storage at the 0- to 5-cm depth in soils from four crop rotations: winter wheat-fallow, spring wheat-chemical fallow, continuous hard red spring wheat, and spring wheat-spring barley on a Ritzville silt loam (Calcidic Haploxeroll). In two incubation studies using 14C-labeled wheat straw, we traced the decomposition of added residue as influenced by (1) cropping frequency, (2) tillage, and (3) pulsed additions of C. Differences in 14C mineralization did not exist among the four rotations at any time throughout the incubations. However, differences in total CO2 production between the continuous wheat rotations and the fallow rotations point to a priming of native SOM, the degree of which appears to be related to the relative contributions of fungi and bacteria to the decomposition of added residue. Addition of non-labeled wheat straw to select samples in the second incubation resulted in a flush of 14C-CO2 not seen in the controls. This priming effect suggests C inputs have a greater effect on mineralization of residual C compared to disturbance and endogenous metabolism appears to be the source of primed C, with priming becoming more pronounced as the fungal:bacterial ratio in the soil increases.