An efficient method for the isolation of mutant antigen-presenting cell (APC) lines is described. When mixtures of transfectant APC lines TAβz (that express Aβz/Aαd MHC class II molecules) and hypothetical variant APC lines TAβd (that express Aβd/Aαd class II molecules) were cultured with and selected by autoreactive Aβz/Aαd-restricted T cell clones, the percentage of TAβd APC lines increased from less than 1% of the original APC mixtures to almost 100% after several cycles of selection. This increase of hypothetical variant was shown to be due to the formation of aggregates of wild-type TAβz APC lines with Aβz/Aαd-restricted autoreactive T cell clones that results in the inhibition of proliferation and probably killing of TAβz APC lines. Based on this, ethyl methane sulfonate (EMS)-treated TAβz APC lines or B-B hybridoma APC lines MW4 (that express Aβz/Aαd and Aβz/Aαz class II molecules) were cultured with and selected by Aβz/Aαd-restricted autoreactive T cell clones to obtain mutant APC lines that escaped the recognition by T cell clones. After cloning, about 43% of clones examined lost the ability to stimulate T cell clones with concomitant loss of class II molecule expression. Less than 1% showed loss of stimulatory activity against T cell clones in spite of the expression of normal amounts of class II molecules. Initial analysis revealed that they include APC mutant lines with (1) altered MHC class II sequences, (2) loss of adhesion molecule expression and (3) possible impairment of the peptide loading. The method described here may provide a variety of mutant APC lines that are useful for the analysis of antigen processing and presentation pathways as well as of class II structure for T cell stimulation.