The spleens of patients with hairy cell leukemia contain high levels of a tartrate-insensitive, cationic, acid phosphatase (the human Type 5 isozyme). This phosphatase has been purified by a procedure which involves only two chromatographic steps: CM-cellulose chromatography and immunoaffinity chromatography on sheep antibodies generated against porcine uteroferrin. Uteroferrin is an abundant iron-containing acid phosphatase that can be recovered readily from porcine uterine secretions. Like uteroferrin, the purified human Type 5 phosphatase is a glycoprotein of molecular weight about 34,000. It contains two atoms of iron/molecule. The human phosphatase and uteroferrin also resemble each other closely in electrophoretic mobility, substrate specificity, and response to a variety of activators and inhibitors. Mouse monoclonal antibodies have been raised to uteroferrin and to the human Type 5 phosphatase. Three monoclonal antibodies which bind with high affinities to distinct sites on the uteroferrin molecule also recognize the human spleen enzyme, but bind to it with much lower affinity. These antibodies also recognize cationic acid phosphatases purified from bovine and rat spleens. A monoclonal antibody raised against the human enzyme, but selected for binding to uteroferrin, appears to recognize a relatively conserved site on all four phosphatases. We conclude that the human Type 5 isozyme belongs to a growing class of structurally related, iron-containing acid phosphatases which includes the iron-transport protein, uteroferrin.