Ammonia production in poultry houses has serious implications for flock health and performance, nutrient value of poultry litter, and energy costs for running poultry operations. The microbial enzyme responsible for final step in the conversion of uric acid (~70% of total N in poultry litter) to ammonia is the urease enzyme. Analysis of ureC (alpha subunit of the urease enzyme) from poultry litter revealed the presence of a novel, dominant group of ureolytic microbes. To target this group, specific primers and probe were designed and a new quantitative real-time PCR assay was developed. The assay allowed for the detection of ³ 1 x 10⁴ ureC target sequences (representing ³ 10 copies per PCR reaction) and amplified target environmental and standard ureC samples with equally high efficiency over 8 orders of magnitude. In comparing a variety of pure cultures and environmental samples known to possess urease activity, target ureC amplification was found only from poultry litter samples, demonstrating the specific nature of this assay. The concentration of cells containing the targeted ureC sequence ranged from 9x10⁶ to 4x10⁸ cells g^-1, and accounted for 0.1 - 3.0% of the total bacterial population in the poultry litter. Different litter types from three different states were analyzed physically, chemically and biologically to determine which environmental parameters most affected the concentration of this targeted ureC group. According to Principal Component Analysis, 40% of the variance in the concentrations of ureC cells was due to the chemical makeup of the litter (organic-N, total N, total C), where as an additional 30% of the variance was accounted for by the physical makeup of the litter (litter composition, moisture content, pH). Future applications of this assay could include determining the efficacy of current ammonia reducing litter amendments or in designing more efficient treatment protocols.