We have sought to determine how much amino acid diversity is tolerable at position 69 of the A* chain, a position previously implicated as a peptlde contact site. Slot-machine mutagenesis was used to create a set of 11 mutant A* cDNAs, each specifying a different amino acid at position 69. These cDNAs were individually expressed In L cells together with a wild-type AJj 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 peptlde to various T hybrldomas was assessed. We found that a surprising degree of amino acid diversity is tolerable at A* position 69: even charged (Glu, Arg) or bulky (Trp, Tyr) residues can be accommodated without abrogating cell-surface expression of A k , 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.