The recognition by cytotoxic T lymphocytes (CTL) of herpes simplex virus (HSV) glycoprotein(s) in association with the H-2K and the H-2D gene products of the H-2b complex was examined by using cell lines derived from H-2 recombinant mice as target cells, and by using H-2 recombinant mouse strains for the generation of HSV-specific CTL populations. CTL from H-2b HSV-immunized mice were found to lyse HSV-infected B6/WT-3 (KbDb) and K5RSV (KbDd) cells, but not KHTGSV (KdDb) cells. Unlabeled HSV-infected K5RSV cells were as efficient in competing for specific CTL lysis of 51Cr labeled HSV-infected B6/WT-3 cells as unlabeled B6/WT-3 cells themselves, whereas infected KHTGSV cells were ineffective. Furthermore, CTL generated in H-2 recombinant mice containing the H-2Kb allele (KbDd) effectively lysed infected B6/WT-3 cells; no specific lysis was observed with immune lymphocytes from those mice containing the H-2Db (KkDb) allele. Limiting dilution analysis of the interleukin 2 (IL 2)-dependent, antigen-independent, CTL precursor populations showed that CTL precursors giving rise to H-2Kb-restricted progeny were present at a relatively high frequency, whereas H-2Db-restricted progeny were present at low frequency or were undetectable. Target cells carrying mutations in the H-2Kb glycoprotein (H-2Kbm1 and H-2Kbm8) infected with HSV-1 were found to be drastically reduced in their ability to be lysed by anti-HSV-1 CTL. HSV-1-infected H-2Kbm5 cells provided a good target for anti-HSV CTL. We conclude that the HSV-specific glycoprotein(s) are recognized by CTL primarily in association with the H-2Kb gene product.