The understandings of phosphorus (P) forms in poultry litter coupled with their transformations in soil contribute to efforts to improve our understanding of long-term P roles in eutrophication. In this study, P in a poultry litter (PL) sample and pasture soil with 20-year PL application, was sequentially extracted into H2O, 0.5 M NaHCO3, 0.1 M NaOH, and 1 M HCl. After appropriate dilution and adjustment to pH 5.0, the fractions were incubated in the presence of orthophosphate-releasing enzymes. Parts of the enzymatically-treated and -untreated fractions were freeze dried for P-31 NMR analysis. NMR analysis demonstrated that the majority of P in PL enzymatically-untreated NaOH and HCl fractions were in organic forms, which was not detected in enzymatically-treated fractions, Thus, these stable organic P forms could be subjected to enzymatic hydrolysis after being applied to soil, which was indeed supported by the soil P data. Compared to non-litter applied soil, 20-year application of PL increases the pools of both labile and stable inorganic P in soil. However, repeated application of PL did not lead to the significant build-up of hydrolyzable organic P in NaOH and HCl fractions, indicating that the stable (hydroxide and acid extractable) organic P must have been degraded to other forms. The degradation of the stable PL organic P observed in this study could be an important mechanism for maintaining a balance between labile and immobile P in soils.