Repeated land applications of poultry litter can change nutrient dynamics in soil. The objectives of this research were to determine if fertilizer type influenced inorganic N availability and resulted in detectable alterations of bacterial community structure. Soil sampling and analysis occurred throughout 2003 and 2004 in small grass plots that have been receiving annual inputs of 2.24 or 8.98 Mg/ha of untreated poultry litter (PL), aluminum sulfate treated poultry litter (AL), equivalent rates (based on N content) of ammonium nitrate (AN), or no amendments (C) since 1995. Soil samples were extracted with 2M KCl and analyzed for ammonium and nitrate. Microbial community structure was assessed using polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE). Nitrate was greatest in soil receiving high rates of AL and AN in both years, with size of pools and relative ratios of nitrate to ammonium changing with year in each treatment. DGGE analysis of 16S rRNA gene fragments detected high numbers of DNA bands, but analysis did not indicate any differences in the Eubacterial communities among treatments. Amplification of amoA gene fragments, which encode for the α subunit of ammonia monooxygenase, resulted in many fewer bands per treatment and in unique patterns among treatments and across years. Changes in bands detected across treatments from 2003 to 2004 may have been related to earlier fertilizer applications and thus earlier sampling dates (cooler and wetter spring samples) in 2004 and differential impact on communities across treatments. Further investigation of changes in specific microbial populations is warranted to improve understanding of how land use practices affect the timing and rates of N cycling.