In the treatment of heart disorders, metoprolol tartrates and digoxin drugs which act by way of blood are used widely. Being vital of using these drug substances, investigation of inhibitory effect on the activities of enzymes in the blood reveals the importance of this work. Aim of this study to investigate effects of metoprolol tartrate and digoxin on human erythrocyte carbonic anhydrase I isoenzyme in vitro conditions. CA-I isoenzyme from human blood has been purified using Sepharose-4B-l-tyrosine-sulfanilamide affinity chromatography method. The enzyme was purified ~116-fold with a yield of 62%. At the end of inhibition studies two substances were showed uncompetitive inhibition. Ki values were determined metoprolol tartrate 5.49±1.86 mM and digoxin 0.0914±0.0062 mM.