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Poultry litter (PL) is a traditionally inexpensive and effective fertilizer to improve soil quality and agricultural productivity. However, over application to soil has raised concern because excess nutrients in runoff could accelerate the eutrophication of fresh water. In this work, we determined the contents of total phosphorus (P), Mehlich 3 extracted P, total nitrogen (N), ammonium (NH4)-N, and nitrate (NO3)-N, in pasture soils receiving annual poultry litter applications of 0, 2.27, 2.27, 3.63, and 1.36 Mg/ha/ yr, respectively, for 0, 5, 10, 15, and 20 years. Samples were collected from three soil depths (0–20, 20–40, and 40–60 cm) of the Hartsells series (fine-loamy, siliceous, subactive, thermic, Typic Hapludults) on a 3–8% slope in the Sand Mountain region of north Alabama. PL application increased levels of total P, Mehlich-3 extractable P, and total N significantly. However, the change in NH4-N and NO3-N contents by the PL application was not statistically significant. Correlation analysis indicated that the contents of total P, Mehlich 3 extracted P, and total N were more related to cumulative amounts of poultry litter applied than the years of application or annual application rates alone. This observation suggested that N and P from poultry litter accumulated in soil. Predicting the build-up based on the cumulative amounts of PL application, rather than isolated factors (i.e., application year or rate), would improve the accuracy of evaluating long-term impacts of poultry litter application on soil nutrient levels.