In this study, carbonic anhydrase (CA: carbonate hydrolyase; E.C.4.2.1.1) was purified from adult Nicotiana tabacum leaves and studied biochemically. The enzyme was purified twice times by using (NH4)(2)SO4 precipation and DEAE-cellulose column chromatography, and its activity was determined for two different substrates (CO2 and p-nitrophenyl acetate). The enzyme obtained from the ion exchange column was purified 40.7 fold and the purity was controlled by 3%-10% discontinuous SDS-PAGE. The pH of the purified enzyme varied between 6.0 and 7.2, the optimum being 6.9. V-max and K-m values were calculated with p-nitrophenyl acetate (0.1524 mM, 0.5446 mM, respectively) as substrate. The optimum temperature for the enzyme was 40 degrees C. The molecular weight of the enzyme and of the subunits were found to be approximately approximate to 137.000 daltons and approximate to 22.800 daltons, respectively. The results indicate that 6 subunits are present. Changes in enzyme activity were determined in the presence of caffeine, nicotine, metal ions and some chemicals.