Continuous monoculture cotton (Gossypium hirsutum) under conventional tillage is the predominant cropping system in the Southern Plains region.  However, other cropping systems and land uses with reduced tillage, higher residue crops, and/or elimination of fallow periods are being evaluated in their potential to increase soil parameters related to soil quality and functioning.  The microbial biomass and community structure, bacterial diversity and enzyme activities of C, N, S and P cycling were studied in a Pullman soil (Fine, mixed, thermic Torrertic Paleustolls; 38% clay and 34% sand) under continuous cotton (Ct-Ct) compared to Conservation Reserve Program (CRP), pasture (Bothriochloa bladhii) and a cotton-corn (Zea mays  L., Ct-Cr) rotation.  Soil microbial biomass C (0-5 cm) was 3 times higher under CRP than under continuous cotton: CRP>Grass=Ct-Cr>Ct-Ct.  Similar trends were found for the activities of glycosidases (CRP=Grass=Ct-Cr>Ct-Ct) and the phosphatases (CRP=grass>Cr-Ct>Ct-Ct).  Comparison of 16S rRNA gene banding pattern and sequences revealed higher number of bacteria species under tilled systems Ct-Ct and Ct-Cr compared to CRP and grass.  Similar number of fatty acids were extracted among systems, but fungal and bacterial indicators were more abundant under CRP and Ct-Cr compared to Ct-Ct.  These findings indicate there are higher number of soil bacteria species under Ct-Ct but in less abundance compared to CRP.  This study demonstrated the potential increases in soil microbial populations and enzyme activities under CRP and pasture and by rotating cotton with a high residue crop (i.e., corn) than continuous monoculture of cotton.