Abstract Hotspot mutations in the p53 tumor suppressor gene result in the disruption of DNA contact points or alter the overall structure of the protein to prevent DNA binding. When inherited, hotspot mutants are associated with Li-Fraumeni syndrome (LFS), a familial cancer predisposition. One of the most common hotspot mutations occurs at codon 175, resulting in an arginine to histidine substitution. We have identified a novel germ line variant of the 175 mutant (Arg to Leu; R175L) in a pediatric patient who developed adrenal cortical carcinoma. Surprisingly, the family is not tumor prone or associated with LFS. In vitro, the R175L mutant displayed an attenuated tumor suppressor activity in the regulation of transcription, colony formation, and apoptosis when compared with wild-type p53 and the R175H mutant. These findings suggest that p53-R175L retains sufficient activity to suppress LFS, but not adrenal cortical carcinoma. Therefore, not all hotspot mutants are functionally equivalent and the biochemical nature of the mutant may significantly influence clinical outcome. The implications of these results for genetic counseling are discussed. (Cancer Res 2006; 66(10): 5056-62)