BALB/c-derived tumor cells were transfected with recombinant Escherichia coli beta-galactosidase (beta-gal) genes which were inserted into IgM heavy chain gene derivatives, leading to expression of the resulting fusion protein in different cellular compartments. A beta-gal-specific, major histocompatibility complex (MHC) class I-restricted CD8+CD4- cytotoxic T lymphocyte (CTL) line of BALB/c origin raised against one transfectant expressing cytoplasmic beta-gal also lysed transfectants expressing beta-gal as membrane-inserted fusion protein, as well as transfectants secreting beta-gal. Our data show that MHC class I-restricted CTL can recognize fragments of nonviral cellular proteins, be they expressed as intracellular, membrane-inserted, or secreted products. The findings confirm and extend a hypothesis on the nature of minor histocompatibility (H) antigens formulated earlier.