Previous research has shown that the addition of flue-gas desulphurization (FGD) gypsum to poultry litter decreases water-soluble P. No information is currently available, however, on extractable P fractions in poultry litter and P availability as affected by gypsum. The first objective of this work was to evaluate the effect of incubation time and rate of gypsum addition to litter alone or litter mixed with soil on total P and inorganic P in sequential extracts of H2O, 0.5 m NaHCO3, 0.1 m NaOH and 1 m HCl. Poultry litter was mixed with 25, 50, or 75% gypsum (by weight) and incubated alone or mixed with soil for 63–93 days at 25 °C, with periodic sequential extractions. For litter alone or litter mixed with soil, adding gypsum decreased total P and inorganic P in the H2O fraction and increased both P forms in the NaHCO3 fraction. These changes did not affect plant P availability as measured by Mehlich-1 P. Increasing incubation time decreased total P and inorganic P in the H2O fraction of litter alone or litter mixed with soil, which was apparently caused by P immobilization by fungi. A second objective of this study was to evaluate P in the H2O and NaHCO3 fractions of litter as affected by stacking time and depth. Litter was stacked to a height of 1.2 m with samples taken immediately after stacking and 31 days later to be sequentially extracted for total P and inorganic P. Stacking time did not affect P in the H2O fraction, but it increased P in the NaHCO3 fraction by 25%. These results suggest that stacking poultry litter may increase the amount of labile P.