Technical Abstract: Poultry manure (PM) contains a large amount of P, and adding this manure to soil can impact the availability of native soil P to plants. To investigate the effects of PM on soil P availability, we grew ryegrass (Lolium perenne) in greenhouse pots amended with poultry manure. Biomass was harvested at 4, 8, and 16 wk following PM application, with soil separated into rhizosphere and bulk fractions. Soil was sequentially extracted by H2O, 0.5 M NaHCO3, 0.1 M NaOH and 1 M HCl, and inorganic P (Pi) and enzymatically hydrolysable organic P (Poe) were quantitated. Root P concentrations were 37% higher and total P uptake 59% higher with PM application. At week 16 there was 30% more labile-Pi in the rhizosphere with PM than in unamended soil. Phosphodiesterase activity increased with PM application. Furthermore, acid phosphomonoesterase, alkaline phosphomonoesterase and phosphodiesterase activities were all higher in the rhizosphere than bulk soil at week 16 with PM, indicating that increased labile-P was due primarily to stimulation of soil phosphatase to mineralize NaOH-Poe. Soil pH increased with PM application and plant growth, and was important in promoting P availability by decreasing sorption of Al- and Fe-associated inorganic and organic phosphates. These results demonstrate that whereas PM application may initially increase NaOH and HCl-Pi, these fractions can be readily changed into labile-P and do not necessarily accumulate as stable or recalcitrant P in soil.