In vitro inhibition effects of indolylchalcones and new pyrido[2,3-d] pyrimidine derivatives on purified human carbonic anhydrase I and II (hCA I and II) were investigated by using CO2 as a substrate. The results showed that all compounds inhibited the hCA I and hCA II enzyme activities. Among all the synthesized compounds, 7e (IC50 = 6.79 mu M) was found to be the most active compound for hCA I inhibitory activity and 5g (IC50 = 7.22 mu M) showed the highest hCA II inhibitory activity. Structure-activity relationships study showed that indolylchalcone derivatives have higher inhibitory activities than pyrido[2,3-d] pyrimidine derivatives on hCA I and hCA II. Additionally, methyl group bonded to uracil ring increases inhibitory activities on both hCA I and hCA II.