To identify new aminothiol radioprotectors that are active when applied topically and have fewer side effects when administered systemically, a new family of aminothiol radioprotectors was designed and synthesized. Three key elements in the aminothiol design were, (1) small size for efficient transmembrane diffusion, (2) positive charged amines in alkyl backbone for strong ionic interaction with DNA backbone, and (3) a perpendicular, alkyl side-chain with a terminal thiol that is projected away from the DNA backbone to enable reactive oxygen species scavenging around DNA. Several in vitro assays were used to characterize the prototype aminothiol, PrC-210, for efficacy: protection against reactive oxygen species-induced plasmid DNA nicking, mass spectrometry to detect aminothiol-reactive oxygen species by-products, S. typhimurium mutagenesis, human cell growth inhibition, Western blot for p21 expression, and FACS analysis. Additionally, two in vivo assays were used to assess radioprotective efficacy; a Sprague-Dawley rat dorsal skin radiodermatitis assay was developed to screen for aminothiol efficacy when topically applied, and ICR mouse survival was scored after systemic PrC-210 administration and whole-body radiation. PrC-210 efficiently scavenged reactive oxygen species and completely protected supercoiled plasmid DNA against reactive oxygen species-induced damage. Neither PrC-210 nor its analog PrC-211 were bacterial mutagens. In cell culture, PrC-210 application to diploid human fibroblasts showed: (1) inhibition of cell growth with an IC(70) of 4.1 mM, (2) induced levels of p21 expression, and (3) a G(1)/S-cell cycle block that was reversed after washout of PrC-210-containing medium. In rodents, PrC-210 was an effective radioprotector showing: (1) complete prevention of Grade 2-3 radiodermatitis when applied topically (370 mM in ethanol:propylene glycol:water solution) prior to skin irradiation, (2) complete prevention of Grade 2-3 radiodermatitis when administered by i.p. injection (200 μg/g of body weight) before skin irradiation, (3) 100% survival of mice from an otherwise 100% lethal dose of whole-body radiation (8.75 Gy) when administered by i.p. injection (252 μg/g of body weight = 0.5 × maximum tolerated dose) before irradiation, and (4) a dose reduction factor of 1.6, the same as amifostine. These data suggest that the PrC-210 aminothiol is a plausible candidate for drug development as a human pre-exposure radioprotector.