In previous work, we observed that H-2k-restricted herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTLs) were effectively able to lyse transfected target cells expressing HSV glycoprotein C (gC), but not cells expressing gB, gD or gE. To confirm and extend our observations on the specificity of anti-HSV CTLs, recombinant adenovirus (Ad) vectors able to express HSV-1 gB or gC (AdgB2 or AdgC) were constructed. Syngeneic target cells infected with AdgB2 were efficiently lysed by primary H-2b and H-2d, but not by H-2k-restricted HSV-specific CTL. Limiting dilution studies indicated that 4 to 10% of H-2b-restricted HSV-specific CTLs recognize gB. H-2k, H-2b and H-2d-restricted anti-HSV-1 CTLs were unable to lyse AdgC-infected syngeneic target cells. To examine the apparent discrepancy between the previous results involving transfected H-2k cells expressing gC and the present results involving AdgC-infected cells, gC-expressing cell lines used in previous experiments were subcloned and retested in CTL assays. DC2 cells which were lysed by HSV-specific CTLs in the previous experiments remained sensitive to anti-HSV CTLs but two other clones derived from the same transfection were not lysed. Further, L cells transfected with the gC or gD gene coupled to the mouse mammary tumour virus promoter and capable of expressing high levels of the glycoproteins following dexamethasone induction were not lysed by H-2k-restricted anti-HSV CTLs. These results suggest that HSV-specific CTLs do not recognize gC, at least when it is expressed using an Ad vector and in most transfected cell lines, whereas a significant proportion of anti-viral CTLs recognize gB presented in some but not all murine haplotypes.