The phospholipid binding activity of cardiotoxin V from Naja naja atra (CTX A5) was studied by use of Langmuir monolayers and found to exhibit pH-dependence in binding to phosphatidylcholine membrane with an apparent pKa around 6.0. Proton NMR investigation of the CTX A5 molecule in the presence of phosphatidylcholine micelles reveals a decrease in association of CTX A5 with membranes at low pH as a result of the protonation of His-4 near the membrane binding site of loop I region of CTX. The pH-dependent binding can be attributed mainly, but not solely, to the change in charge content of the CTX molecule upon His-4 protonation at the membrane/water interface. This is shown by analyzing the pH- and ionic strength dependence of binding of CTXs to phospholipid monolayers according to Gouy-Chapman theory. The protonation of the His-4 residue also results in a local conformational change in the loop I region since the chemical shifts of amide protons for the amino acid residues from Cys-3 to Thr-14 are all found to vary as a function of pH with an apparent pKa similar to that of His-4. Interestingly, the effect is relayed to other amino acid residues in the structural core of the protein such as those in C-terminal (Lys-60, Cys-61, and Asn-62) and triple-stranded antiparallel beta-sheet (Cys-22, Lys-24, Ala-25, Arg-38, and Ala-41) regions. An additional local conformational change in the molecule results around pH 5 as evidenced by circular dichroism spectroscopic studies, although this change does not affect the characteristic beta-sheet and three-finger loop structure of CTX molecule as revealed by two-dimensional NOESY 1H NMR study. The latter conformational change at acidic pH, however, completely inactivates CTX-induced aggregation/fusion activity of sphingomyelin vesicles. The results suggest that deciphering the functional sites of CTXs on the basis of structure and dynamics determined at low pH should be done with caution. Since 19 out of 44 CTX homologues with known amino acid sequence contain His-4, the effect of His-4 on the structure and function of CTX molecules is important and is discussed in terms of the diverse membrane targets of CTX subtypes. Also discussed is the pH-induced activation of snake venom proteins in the victim.