Peptide hydrolases (EC 3.4.-.-) were solubilized from purified brush borders of rat intestinal mucosa by papain digestion. Three peptide hydrolases, I, II, and III, with different substrate specificities were isolated by means of DEAE-cellulose chromatography and preparative acrylamide gel electrophoresis. On repeat preparative acrylamide gel electrophoresis under slightly different conditions, enzyme II was resolved into two proteins, IIa and IIb, with vary similar, possibly identical, substrate specificities. Efforts to discover additional brush border peptide hydrolases revealed none. Studies using more than 50 substrates showed that enzyme I was most active against Met-Met, Met-Ala, and Met-Phe while enzyme II was most active against Phe-Gly, Phe-Ser, and Leu-Gly-Gly, and enzyme III most rapidly hydrolyzed Gly-Leu, Leu-Gly, and Met-Gly. Efforts to discover substrates which are highly discriminating for each enzyme were partly successful. Thus, a number of substrates including leucine amide, leucyl-beta-naphthylamide and Phe-Asp were hydrolyzed almost exclusively (95% or more) by enzyme II while Gly-Leu was similarly specific for enzyme III. No substrate highly discriminating for enzyme I was discovered. Ion-exchange chromatography resulted in increases in specific activity of 10- and 120-fold for enzymes II and III, respectively. By sequential use of ion-exchange chromatography and preparative acrylamide gel electrophoresis, each of the three enzymes was partially purified to the point that they were free of contaminating disaccharidases and enzymes I and II gave single dense bands on analytical acrylamide gel electrophoresis while enzyme III gave a single dense band plus one additional faint protein band. Under appropriate conditions, analytical gel electrophoresis also resolved enzyme II into two bands with enzyme activity. The three enzymes were isolated from intestinal brush borders of germ-free rats indicating that none of the enzymes is of bacterial origin. With Phe-Gly as substrate, pH optima for enzymes I, II, and III were 8.0, 8.0, and 8.5, respectively. Molecular weights determined by gel filtration were 283 000, 284 000, and 134 000, respectively. Studies of activation by metal ions and inhibition by metal ion chelators suggested that the activity of each of the enzymes is dependent on a relatively tightly bound metal cofactor. Peptide hydrolases of the intestinal mucosa play an essential role in protein digestion. The studies presented here help to clarify the total number and substrate specificities of these enzymes in the rat brush border.