Poultry manure (PM) contains a large proportion of phosphorus (P) in mineral-associated forms that may not be readily available for plant uptake. In addition, PM application influences both chemical and biotic processes, and can affect the lability of native soil P. 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 weeks 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 hydrolyzable organic P (Poe) were quantitated. Root P concentrations were 37% higher and total P uptake 59% higher with PM application than Control. At week 16, there was 30% more labile-Pi (H2O- plus NaHCO3-Pi) in the rhizosphere with PM than in Control. Phosphodiesterase activity increased with PM application. Furthermore, acid phosphomonoesterase, alkaline phosphomonoesterase, and phosphodiesterase activities were all higher in the rhizosphere than in bulk soil at week 16 with PM, indicating that increased labile-Pi was due primarily to stimulation of soil phosphatases to mineralize NaOH-Poe. Soil pH increased with PM application and plant growth, and may have promoted 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.