Alkaline phoshatase (orthophosphoric monoester phosphohydrolase EC 3.1.3.1) which hydrolyzes a variety of monophosphate esters was purified from rat uterus, using butanol extraction, acetone precipitation, gel filtration on Sephadex G-150, affinity chromatography on Concanavalin A-Sepharose 4B, and isoelectric focusing. This enzyme has the molecular weight of 560,000 daltons by gel filtration and it is very unstable below pH 6.0 and the most stable between pH 8-9. This enzyme shows pH optimum shift of 0.62 pH with 10-fold increase in substrate concentration. Ionic strength in the reaction buffer affects the enzyme activity that is, increase in ionic strength decreases Km and increases the V_(max). The log V_(max) is primarily dependent on the square root of ionic strength. From the thermostability study, it may be concluded that alkaline phosphatase from rat uterus is a different isoenzyme from that of other rat tissues like placenta, liver, and intestine. Among divalent cation, Mg^(2+) is a good activator for the enzyme and Ca^(2+) shows an activation only when its concentration is more than 100 mM. The optimum pH of enzyme activation by Mg^(2+) does not coincide with the optimum pH of enzyme activity. This enzyme has a multiple pI values of pI 6.5-7.1 from the isoelectric forcusing. This enzyme strongly binds to DEAE-Sephadex not by the normal charge to charge interaction but by an uncertain interaction between the enzyme and DEAE-ligand. The bound enzyme activity is not eluted even with the high ionic strength buffer containing 1.2 M NaCl. But it can be eluted by the 1 M solution of 2-amino-2-methyl-1, 3-propanediol.