Bothropstoxin-I (BthTx-I) is a homodimeric Lys49-PLA₂ present in the venom of the snake Bothrops jararacussu. Although lacking hydrolytic activity against phospholipid substrates, BthTx-I damages artificial membranes by a Ca²⁺-independent mechanism. We have previously suggested that a transition between "open" and "closed" membrane bound dimer conformations is responsible for this activity. In order to study the interaction of the BthTx-I with model membranes by intrinsic tryptophan (Trp) fluorescence spectroscopy, the single Trp77 residue at the dimer interface was substituted by histidine (W77H). A second round of mutagenesis 9 separate Trp mutants were produced to create double mutants each with a unique Trp residue located either in the dimer interface (at positions 7 and 10), dorsal surface (at positions 46 and 104), ventral surface (at positions 31 and 67) or in the C-terminal loop region (at positions 117,119 and 125). After expression of the mutant genes in E. coli using the pET3d vector, the recombinant proteins were refolded and purified by cation-exchange chromatography, after which circular dichroism spectroscopy demonstrated that native-like secondary structure of the protein was maintained. The damaging activity of these mutants against unilamellar liposomes was correlated with the changes in the intrinsic tryptophan emission spectra in presence and absence of the liposomes. The affinity of the mutants for the membranes was estimated by ultracentifugation coupled with analysis of the pellet and supernatant fractions by SDS-PAGE. The results demonstrated that the affinity and membrane damaging activity was influenced by the degree of negative charge of the liposome membrane. These changes could be correlated with alterations in the microenvironments of each Trp when associated with the liposomes. These results permit an evaluation of the local protein conformation changes during the Ca²⁺-independent membrane damaging process.