Although it has been demonstrated that CTLs can be raised against tumor-associated self-antigens, achieving consistent and effective clinical responses has proven challenging. Superagonist altered peptide ligands (APLs) can often elicit potent antitumor CTL responses where the native tumor-associated epitope fails. Current methods have identified a limited number of superagonist APLs, including the prototypic 27L mutant of MART-1. However, more comprehensive screening strategies would be desirable. In this study, we use a novel genetic screen, involving recombinant technology and class I Ag cross-presentation, to search for supraoptimal superagonists of the 27L MART-1 mutant by surveying the effectiveness of virtually every single amino acid substitution mutant of 27L to activate human Ag-specific CTL clones recognizing the wild-type MART-1(26-35) epitope. We identify three novel mutant epitopes with superagonist properties that are functionally superior to 27L; however, the ability of a given analogue to act as superagonist varies among patients and suggests that a given superagonist APL may be ideally suited to different patients. These findings endorse the use of comprehensive methods to establish panels of potential superagonist APLs to individualize tumor peptide vaccines among patients.