Carbonic anhydrase II (CA II) deficiency has been shown to be the primary defect in the recessively inherited syndrome of osteopetrosis with renal tubular acidosis. Until now, the absence of CA II in kidney of CA II-deficient patients has not been shown directly, and the status of the membrane-associated CA in kidney of CA II-deficient patients has been unclear. To address these questions, we analyzed urinary membranes and soluble fractions from normal and CA II-deficient subjects. The CA activity in membrane fractions of normal urine was found to comprise two components--(i) a vesicle-enclosed, sodium dodecyl sulfate (SDS)-sensitive fraction, which was shown immunochemically to be the 29-kDa CA II, and (ii) an SDS-resistant fraction, which was due to native and cleaved forms of the 35-kDa, membrane-anchored isozyme CA IV. Urinary membranes from CA II-deficient patients showed little or no SDS-sensitive activity and no immunoreactivity for CA II, providing direct evidence that their mutation, which produces CA II deficiency in erythrocytes, also affects CA II in kidney. CA IV activity and immunoreactivity were present in normal amounts in urinary membranes from CA II-deficient patients. We conclude from the enzymatic and immunological evidence presented that both CA II and CA IV are present in urinary membranes from normal subjects, that renal CA IV is present but renal CA II is absent in urinary membranes from patients with the CA II-deficiency syndrome, and that the methods presented should be useful in studying renal CA II and renal CA IV in other disorders of impaired bicarbonate reabsorption.