Soil organic matter is important both from an agronomic and an environmental perspective because it affects the capacity of the soil to sustain crop growth, and it is a source and sink of atmospheric CO2-C. Soil microbial biomass comprises a small proportion of total soil organic matter, but it is more dynamic than total soil organic matter. Therefore, measurements of soil microbial biomass may show the effects of soil management on potential changes in soil organic matter before such effects can be detected by measuring total soil organic matter. The effects of tillage and crop rotation on soil microbial biomass and activity were studied in 1995–1997 in the wheat phase of different cropping rotations that had been established in 1992 under zero tillage or conventional tillage in northern Alberta. Soil microbial biomass was often significantly (P < 0.05) higher, but never significantly lower, under zero tillage than under conventional tillage. However, CO2 evolution (basal respiration) was usually higher under conventional tillage than under zero tillage, resulting in higher specific respiration (qCO2) under conventional tillage than under zero tillage. The higher additions but lower losses of labile C under zero tillage mean that more C is sequestered in the soil in the zero-tillage system. Thus, this system contributes less to atmospheric CO2 than conventional tillage, and that soil organic matter accumulates more under zero tillage. Plots preceded by summerfallow, especially under conventional tillage, usually had the lowest microbial biomass and CO2 evolution, and plots preceded by legume crops had higher microbial biomass and lower qCO2 than other treatments. Tillage and rotation had little effect on total soil organic matter 5 yr after the treatments had been imposed, probably because of the cold climate of northern Alberta, but the results confirm that the labile forms of soil C are more sensitive indicators of soil organic C trends than total soil organic C. These effects of tillage and rotation on soil microbial biomass were similar to those on microbial diversity reported previously. These results confirm that zero tillage and legume-based crop rotations are more sustainable crop management systems than conventional tillage and fallowing in the Gray Luvisolic soils of northern Alberta. Key words: Carbon sequestration, carbon mineralization, microbial activity, soil organic matter