Beef feedyards produce nitrous oxide (N2O), a potent greenhouse gas. Limited research has evaluated the processes that produce feedyard N2O, and how rainfall and temperature impact N2O losses. Manure in feedyard pens develops into a complex ecosystem of microbes, extracellular enzymes, feces, and urine, with varying H2O content. This study aimed to improve understanding of feedyard N cycling under differing environmental conditions by incubation of manure in simulated feedyard pens using large chambers under laboratory conditions. We hypothesized that nitrification was the primary source of feedyard N2O, with interactions among temperature, H2O content, and manure properties. Emissions of N2O were monitored with a realtime N2O analyzer. Manure samples were taken at intervals for analyses of physicochemical properties, denitrification enzyme activity (DEA), and nitrification activity (NA). Due to equipment limitations, there was only one chamber per temperature tested. Correlation was poor among N2O emissions and rates of DEA and NA. However, significant relationships were found among key manure characteristics, such as ammonia/ammonium and nitrate/nitrite concentrations, manure dry matter, redox status, and temperature. These data suggest that most N2O was derived from denitrification in the top 5 cm of the manure pack. Further study is warranted to identify the processes involved in flushes of N2O emitted immediately after rainfall, possibly due to abiotic chemical reactions that release N2O sequestered in manure pores.