A new variant of human erythrocyte carbonic anhydrase II (CAII) was discovered in a single heterozygous individual during routine screening of blood samples from the island of Java in Indonesia. The normal and variant components of the heterozygous CAII mixture were resolved by isoelectric focusing following purification by a specific affinity matrix. Specific esterase activities and Michaelis-Menten constants were identical. Only very small differences were noted with respect to inhibition by acetazolamide and chloride. Double diffusion analysis showed the immunological identify of the normal and variant enzymes. The variant CAII was considerably less heat stable than the normal enzyme. The variant was slightly more stable than the normal enzyme upon dialysis against the zinc chelator dipicolinic acid (PDCA), indicating a tighter binding of zinc than the normal enzyme. Analysis of tryptic peptides from the normal and variant enzymes indicated that, in the variant, lysine at position 17 from the N terminus had changed to glutamic acid. The differences in physiochemical properties observed for the normal and variant enzyme are discussed in relation to the possible effects of this substitution on the structure of the CAII molecule.