The chloroplast transit peptide (cTP) of pea carbonic anhydrase was shown to be processed at two different sites, giving protein subunits of two sizes. The cleavage sites were identified and found to be localized immediately before and after a highly charged part, containing 8 acidic and 6 basic residues, of the cTP. Properties of pea carbonic anhydrase produced in Escherichia coli show that folding, oligomerization and catalytic activity do not depend on the presence of the acidic part or the rest of the cTP. The pattern of processing of the cTP in E. coli indicates that cleavage at site I is specific for a chloroplastic stromal peptidase and that cleavage at site I prevents processing at site II.