Soil organic matter dynamics and composition reflect the effects of vegetation, biota, climate, physical protection, and land-use. In the short-term we might expect the decomposition of litter and the stable organic matter to be most affected by the size and composition of the microbial community and the chemistry of the substrate. We compared soils from two sites, Colorado and Ohio, to see how litter and soil organic matter decomposition was affected by microbial biomass size and the amount and characteristics of the soil organic matter of these soils. Both sites had native, minimal cultivation, and conventional tillage treatments with the Colorado site having greater sand content and lower soil organic C (SOC) (0.7-1.2%) and the Ohio site having higher clay content and higher SOC (1.8-4.7%). We added corn or wheat litter, with different 13C contents, to soils that were recently sampled and soils that had been incubated in the lab for over 700 days and measured the 13CO2 respired and size of the microbial biomass during a 42 day incubation. The previously incubated soils had lost between 10-30% of their original SOC and the microbial biomass had declined by 41-64% prior to the substrate addition. Recently sampled soils respired between 31 to 46% of the litter added. Native soils, which had the greatest SOC and microbial biomass, respired the least amount of litter. In contrast, the minimally cultivation soils at both sites respired greater amounts of litter even though they had the lowest microbial biomass. The soils incubated for over 700 days, which were more depleted in labile SOC, gave variable results. Some soils respiring more and some less litter than their recently sampled counterparts. This may mean the microbial community composition remaining after long-incubation and its regrowth rate has a greater influence on litter decomposition than the size of the microbial biomass.