The cysteine proteinase B of Leishmania parasites is an important virulence factor. In this study we have expressed, isolated and characterized for the first time a recombinant CPB from Leishmania braziliensis, the causative agent of mucocutaneous leishmaniosis. The mature region of the recombinant CPB shares a high percentage identity with its Leishmania mexicana CPB2.8 (rCPB2.8ΔCTE) counterpart (76.36%) and has identical amino acid residues at the S₁, catalytic triad and S^′₁ subsites. Nevertheless, when the kinetics of substrate hydrolysis was measured using a combinatorial library of internally quenched fluorescent peptides based upon the lead sequence Abz-KLRSSKQ-EDDnp, significant differences were obtained. These results suggest that the differences in substrate utilization observed between the L. mexicana and L. braziliensis CPs must be related to amino acid modifications outside the core of the active site cleft. Moreover, a potent inhibitor with Pro at P1 and high affinity for L. braziliensis recombinant CPB showed less affinity to L. mexicana CPB 2.8, which preferred Phe, Leu, and Asn at the same position.