Charge discrimination in P2X receptors occurs in two stages. The first stage takes place in the extracellular vestibule. The second one happens as the ions travel across the pore. The search of the amino acids required to achieve these goals has focused on negatively charged residues conserved among the family members. This strategy, however, has afforded baffling results since residues that strongly influence ion selectivity in a given member are not present in others. This finding suggests that alternative family members could achieve the same goal using different molecular approaches. We have compared the mechanisms of charge discrimination in the extracellular vestibule of zebrafish P2X4 (zfP2X4) and human P2X3 (hP2X3), employing molecular dynamics simulations. In particular, we have analyzed how the mutation of residues D59 and D61 of zfP2X4 and residues E46, D53, and E57 of hP2X3 influence ion behavior. The results indicate that both D59 and D61 are required to confer the extracellular vestibule of zfP2X4 a preference for cations. In contrast, the presence of D53 suffices to provide that capacity to hP2X3. We also computed the potentials of mean force for the passage of Na and Cl through the pore of hP2X3. These profiles were compared against those already available for zfP2X4. Altogether, the results provide a detailed description of the mechanisms employed by these receptors to discriminate between cations and anions.