Cationic amino acids contribute to alpha-defensin bactericidal activity. Curiously, although Arg and Lys have equivalent electropositive charges at neutral pH, alpha-defensins contain an average of nine Arg residues per Lys residue. To investigate the role of high alpha-defensin Arg content, all Arg residues in mouse Paneth cell alpha-defensin cryptdin 4 (Crp4) and rhesus myeloid alpha-defensin 4 (RMAD-4) were replaced with Lys to prepare (R/K)-Crp4 and (R/K)-RMAD-4, respectively. Lys-for-Arg replacements in Crp4 attenuated bactericidal activity and slowed the kinetics of Escherichia coli ML35 cell permeabilization, and (R/K)-Crp4 required longer exposure times to reduce E. coli cell survival. In marked contrast, Lys substitutions in RMAD-4 improved microbicidal activity against certain bacteria and permeabilized E. coli more effectively. Therefore, Arg-->Lys substitutions attenuated activity in Crp4 but not in RMAD-4, and the functional consequences of Arg-->Lys replacements in alpha-defensins are dependent on the peptide primary structure. In addition, the bactericidal effects of (R/K)-Crp4 and (R/K)-RMAD-4 were more sensitive to inhibition by NaCl than those of the native peptides, suggesting that the high Arg content of alpha-defensins may be under selection to confer superior microbicidal function under physiologic conditions.