To study the relationship between the structure and function of Ia antigens, as well as the physiologic requirements for antigen presentation to major histocompatibility complex-restricted T cells, class II A alpha and A beta genes from the k and d haplotypes were transfected into Ltk- fibroblasts using the calcium phosphate coprecipitation technique. Individually transfected genes were actively transcribed in the L cells without covalent linkage to, or cotransformation with, viral enhancer sequences. However, cell surface expression of detectable I-A required the presence of transfected A alpha dA beta d or A alpha kA beta k pairs in a single cell. The level of I-A expression under these conditions was 1/5-1/10 that of Ia+ B lymphoma cells, or B lymphoma cells expressing transfected class II genes. These I-A-expressing transfectants were tested for accessory cell function and shown to present polypeptide and complex protein antigens to T cell clones and hybridomas in the context of the transfected gene products. One T cell clone, restricted to I-Ak plus GAT (L-glutamic acid60-L-alanine30-L-tyrosine10), had a profound cytotoxic effect on I-Ak- but not I-Ad-expressing transfectants in the presence of specific antigen. Assays of unprimed T cells showed that both Ia+ and Ia- L cells could serve as accessory cells for concanavalin A-induced proliferative responses. These data indicate that L cells can transcribe, translate, and express transfected class II genes and that such I-A-bearing L cells possess the necessary metabolic mechanisms for presenting these antigens to T lymphocytes in the context of their I-A molecules.