Some of the acid phosphatase isozymes of bovine leucocytes and plasma have been separated and partly characterized. About 80% of the phosphatase activity of leucocytes at pH 4·9 was particle-bound and about 8% was extractable with Amberlite CG-50 ion exchange resin. This extractable enzyme existed as a single electrophoretic component with a mol. wt of about 42000 and with optimum activity at pH 5·8. Km for p-nitrophenyl phosphate was 1·6 mM at pH 5·8 and 0·4 mM at pH 4·9. At pH 5·8 orthophosphate (K1 = 1·5 mM) and pyrophosphate (Ki = 4·1 mM) were competitive inhibitors. The enzyme was also strongly inhibited by F−, Al3+, IO4− and S2032−. The enzyme which was not extractable with Amberlite was very heterogeneous with respect to molecular weight. At the pH optimum (4·9), Km for p-nitrophenyl phosphate was 0·4 mM and orthophosphate (K1 = 2·3 mM) and pyrophosphate (K1 = 2·1 mM) were competitive inhibitors. Other inhibitors included F−, Al3+, Hg2+, IO4− and tartrate. The enzyme extracted from plasma by Amberlite CG-50 treatment had properties similar to that extracted from leucocytes. Normal bovine milk contained a single acid phosphatase, but milk from cows with mastitis showed 3 electrophoretic isozyme bands, one being the same as in normal milk; the 2 additional bands were of leucocyte origin.