Rosmarinic acid is a water-soluble ester of caffeic acid and 3,4-dihydroxyphenyllactic acids, and is mainly found in plant species including Boraginaceae and Lamiaceae. In this research, we determined the inhibition property of rosmarinic acid on carbonic anhydrase isoenzymes I and II (hCA I and II) purified from human erythrocytes by using Sepharose-4B affinity column chromatography. hCA I and II isoenzymes were obtained with a yield of 57.9% and 67.2% and 76.5- and 509.3-fold purification of each isoenzyme, respectively. In order to show the purity of the isoenzymes, SDS-PAGE was performed and one band was observed. In vitro inhibition of both hCA I and II isoenzymes by rosmarinic acid using CO2-esterase activity gave Ki values of 86.0 mu M and 57.0 mu M, respectively.