Human kidney alanine aminopeptidase was purified to apparent homogeneity as judged by electrophoresis and sedimentation in the analytical ultracentrifuge. Amino acid analyses indicate that the enzyme is high in tryptophan content and low in cysteine content. The enzyme contains sialic acid, hexoses and glucosamine, which make up 21% of its dry wt. In dilute buffer, the enzyme exhibits a MW near 236,000, but in denaturing solvents the enzyme exhibits a MW near 119,500. Zn analyses by atomic absorption demonstrate 1 mol of Zn for 113,500 .+-. 6900 g of protein. The Zn is firmly bound, since exhaustive dialysis against chelating agents does not remove the Zn or inactivate the enzyme. The enzyme is stimualted by Co2+ 1.65-fold, but, in contrast to the enzyme-Zn complex, the enzyme-Co complex dissociates upon dialysis. Kinetic studies with a series of aminoacyl-.beta.-naphthylamides indicated that the highest kcat value was obtained for L-alanyl-.beta.-naphthylamide (8.43 .times. 103 s-1), whereas the lowest Km value was obtained for L-methionyl-.beta.-naphthylamide (1.4 .times. 10-5 M).