Cultivation is known to influence the organic matter status and structural stability of soil. We investigated the effects of 69 yr of cultivation on the nature, distribution and activity of microbial biomass (MB) in different aggregate size classes of an Orthic Brown Chernozemic soil. Cultivation decreased MB content, its activity and enzyme activity in soil. Microaggregate (<0.25mm) size classes in both native and cultivated soils contained lower organic-C, MB-C, fungal biomass, arylsulfatase, acid phosphatase and respiratory activities as compared to macroaggregates. However, the negative effects of cultivation were more pronounced on macroaggregate size classes. Nutrient ratios of both whole aggregates and microbial biomass were narrower in aggregates from cultivated soil as compared to native soil. In both native and cultivated soils, mineralization of C. N and S was greater in macroaggregates as compared to that in microaggregates. The greatest effect of cultivation on nutrient and microbial characteristics was observed in the 0.25 to 1.00 mm dia size classes. These results suggest that microbial biomass, especially fungal biomass, plays an important role in the formation of macroaggregates and is the labile organic matter that serves as the primary source of C and nutrients released following cultivation.