Due to their involvement in diverse pathological conditions, carbonic anhydrases have been the targets of drug developments for the treatments of glaucoma, epilepsy, high altitude sickness, as well as cancer. Of about 14 isozymes of carbonic anhydrases, carbonic anhydrase-II (hCA-II) has been most extensively investigated from the structural, functional, and inhibitor design point of view. We discovered that hCA-II preferentially binds to the Sepharose-iminodiacetate (IDA)-Zn(2+) column, and such binding does not require incorporation of either N- or C-terminal histidine tags in the protein structure. By using the Sepharose-IDA-Zn(2+) affinity column, we purified the Escherichia coli expressed hCA-II with an overall recovery of 76%. The purified enzyme showed a single band on the SDS-PAGE. Due to ease in preparing the Sepharose-IDA-Zn(2+) column, and purifying hCA-II just in one step, the overall protocol will be ideal for producing bulk quantities of the enzyme for high throughput screening of inhibitors.