Activities of phytase, a pH 6.0 optimum nonspecific phosphomonoesterase and phosphodiesterase assayed toward bis(p-nitrophenyl)phosphate (phosphodiesterase I) and against p-nitrophenylphosphorylcholine (phosphodiesterase II), were partially purified from mycelial extracts of Aspergillus niger AbZ4 cultivated on a molasses medium by a liquid surface fermentation method. After elimination of phosphate from the medium, 7.3- and 3.5-fold enhancements in specific activities of phytase and phosphodiesterase II were observed. Efficacies of mycelial protein fractions in dephosphorylating a wheat-based broiler feed were determined in vitro according to a procedure that simulated digestion in the intestinal tract of poultry. The addition of 0.052 mg of protein from fractions, each of which was high in either pH 6.0 optimum phosphomonoesterase, phosphodiesterase I, phosphodiesterase II, or phytase per gram of a feed sample resulted in the enhancement of phosphorus release by 10, 11, 27, and 88%, respectively. In the presence of an excess of commercial phytase, the addition of the mycelial fraction high in phytase increased the dephosphorylation rate by 56%. The fraction high in phosphodiesterase II enhanced feed dephosphorylation by 8% in the presence of an excess of commercial phytase and commercial acid phosphatase.