Technical Abstract: Repeated additions of untreated and aluminum sulfate (alum)-treated poultry litter to soil affect ecology and consequent nutrient dynamics. The objective of this study was to determine how repeated annual poultry litter additions affected phosphatase activities in concert with changes in soil phosphorus (P). Field plots were amended annually since 1995 with either 2.24 or 8.96 Mg/ha of alum-treated (AL-1 or AL-4, respectively) or untreated poultry litter (PL-1 or PL-4, respectively) or equivalent rates by N content of ammonium nitrate (AN-1 or AN-4, respectively). Soil pH, total C (CTot), microbial biomass C, double-stranded deoxyribonucleic acid (dsDNA), Mehlich-III P (M3P), water-soluble P (WSP), and acid and alkaline phosphatase activities were measured before and 10 days, 1 month, and 6 months after fertilizer applications in 2003 and 2004. M3P and acid phosphatase activities were higher in AL-4 soil than in all other treatments. Higher WSP in the untreated compared to the alum-treated litter resulted in higher WSP in the soil amended with untreated litter. At the same time, alkaline phosphatase activities were significantly higher in PL-4 compared to AL-4. In contrast, alkaline phosphatase activities were significantly lower in AN-4, the treatment with the lowest pH. Additionally, alkaline phosphatase activities expressed per unit CTot or dsDNA remained significantly greater in PL-4 and significantly lower in AN-4, than all other treatments. Thus, some factor beyond an increase in soil C or microbial biomass contributed to elevated alkaline phosphatase activities in PL-4, despite increased WSP in the treatment receiving the high rate of untreated litter.