Land application is the most common and usually most desirable method of utilizing manure because of nutrient and organic matter addition to soils. In recent years, as a result of the shift to fewer and larger confined animal operations, environmental and economic issues associated with utilization or disposal of animal manures and litters has become a focal point of conservation efforts. The potential excessive nutrient and/or microbial loading from mismanaged land-applied organic fertilizers has drawn increased media, public, and regulatory attention forcing changes in animal waste management. Currently, it is not clear to what extent different rates of poultry litter impact soil microbial communities. These communities control nutrient availability, organic matter quality and quantity, and soil degradation potential, and thus, control soil quality and functioning. From 2002 to 2004, this study investigated microbial community shifts and several enzyme activities in a Vertisol soil (Houston Black Clay soil; Fine, smectitic, thermic, Udic Haplustert) at 0-15 cm as affected by different rates of poultry litter application to pasture (0, 6.7, and 13.4 Mg ha-1) and cultivated sites (0, 4.5, 6.7, 9.0, 11.2, and 13.4 Mg ha-1) in Texas, USA. No differences in soil C content (pasture: 4.39± 0.32% or cultivated: 4.02± 0.67%) or pH (avg: 7.95) were observed following the first four years of litter application. Compared to the control sites with no applied litter, microbial biomass C and N increased at litter rates greater than 6.7 Mg ha-1. Litter rates of 13.4 Mg ha-1 to cultivated sites caused increases in the soil MBC or MBN comparable to the levels observed in non-treated pasture sites in 2003. Beta-glucosidase, beta-glucosaminidase, and alpha-galactosidase activities increased at litter rates greater than 6.7 Mg ha-1. Alkaline phosphatase and arylsulfatase activities increased at litter rates of 6.7 Mg ha-1 and higher in pasture, and at the 11.2 and 13.4 Mg ha-1 application rates in cultivated sites. According to Principal Component Analysis of fatty acids methyl ester profiles, the microbial community structure under pasture experienced shifts to higher soil bacterial populations at litter rates of 6.7 Mg ha-1, and there were shifts to higher soil fungal populations at litter rates of 9.0, 11.2, and 13.4 Mg ha-1 in cultivated sites. While our study demonstrated that rates greater than 6.7 Mg ha-1 provided rapid enhancement of soil microbial populations and enzymatic activities, they result in P application in excess of crop needs, which increases soil P levels and the potential for excess P loss in runoff. Thus, studies will continue to investigate if litter application at rates below 6.7 Mg ha-1, previously recommended to maintain water quality, will result in similar improved soil microbial and biochemical properties with continued annual litter application.