There are three known groups of human Ia antigens; HLA-DR, HLA-DQ (formerly DC or DS), and HLA-DP (formerly SB). This study investigates whether gene complementation operates in the expression of human Ia antigens. Epstein Barr Virus (EBV) lymphoblastoid cell lines were produced from the peripheral blood mononuclear cells of a family unit (both parents and a child). Cytotoxic T cell (CTL) clones were raised against the EBV lymphoblastoid cell line of the child utilizing lymphocytes from an unrelated donor. These clones were then screened for activity against the EBV lymphoblastoid cell lines derived from both parents. Two stable CTL clones were identified that exhibited CTL activity predominantly against the child and not against either parent. Both clones were T3+, T4+, T8-, DR+, DQ+. The activity of one of the clones was completely blocked by anti-HLA-DQ reagents, but not by an anti-class-I, or an anti-HLA-DR reagent. The other clone was blocked by a polyspecific anti-Ia reagent but not by an anti-class-I, anti-HLA-DR, or anti-HLA-DQ reagent. These studies suggest that gene complementation plays a role in human Ia antigen expression, producing hybrid Ia antigens in a child that are not present in either parent.