AmyI-1-18, an antimicrobial peptide, is a cationic -helical octadecapeptide derived from -amylase of rice (Oryza sativa L. japonica) that contains four cationic amino acid residues (two arginines and two lysines). To enhance the antifungal activity of AmyI-1-18 against Candida albicans, 11 analogs bearing substitutions with alanine, leucine, and/or arginine, which were designed on the basis of the helical wheel projection of AmyI-1-18, were synthesized, and their antifungal activity was investigated. The antifungal activities of four analogs obtained by replacing arginine or lysine with alanine were significantly reduced. The results suggested that the cationic arginine and lysine residues in AmyI-1-18 are important for its antifungal activity. The antifungal activities of two single leucine-substituted analogs were not improved, but among three single arginine-substituted analogs, AmyI-1-18(D15R) had approximately a twofold higher antifungal activity [50% growth-inhibitory concentration (IC50): 31 M] than AmyI-1-18 (IC50: 64 M) and exhibited low hemolytic activity (4% at 100 M). Flow cytometric analysis using propidium iodide revealed that the antifungal activity of AmyI-1-18(D15R) was dependent on its membrane-disrupting activity in a manner different from that of AmyI-1-18. Further enhancement of the cationicity and hydrophobicity of AmyI-1-18(D15R) resulted in no improvement in antifungal activity and a significant increase in hemolytic activity. In this study, the results demonstrated that the antifungal activity of AmyI-1-18 against C. albicans was enhanced through increasing its membrane-disrupting activity by replacing aspartic acid at position 15 with arginine without a significant increase in hemolytic activity. (c) 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 219-229, 2016.