We have sought to determine how much amino acid diversity is tolerable at position 69 of the Akα chain, a position previously implicated as a peptide contact site. Slot-machine mutagenesis was used to create a set of 11 mutant Akα cDNAs, each specifying a different amino acid at position 69. These cDNAswere individually expressed In L cells together with a wild-type Akβ cDNA to produce a panel of mutant antigen-presenting cell lines. The ability of each member of this panel to present a hen egg lysozyme and a bovine ribonuciease peptide to various T hybridomas was assessed. We found that a surprising degree of amino acid diversity is tolerable at Akα position 69: even charged (Glu, Arg) or bulky (Trp, Tyr) residues can be accommodated without abrogating cell-surface expression of Ak, peptide binding to it, or T cell recognition of it. We discuss the Implications of these findings for models of T cell recognition of the class II molecule/antigen duplex.