Beta-lactams use aqueous channels of porins to penetrate Gram-negative bacteria. The L3 loop of Escherichia coli OmpF porin is a key feature that actively contributes to both channel size and electrostatic properties. Acid residues D113, E117, and D121 are responsible for the negative part of the local electrostatic field on this loop. Two substitutions, D113A and D121A, located in the negatively charged cluster of the OmpF eyelet, increase the likelihood of producing bacteria susceptible to several beta-lactams. D113A substitution results in an increase in the ampicillin, cefoxitin, and ceftazidime susceptibility. Molecular modeling suggests that the charges harbored by the beta-lactam molecules interact with the charged residues located inside the porin eyelet.