Based on in vivo studies, it has been suggested that the extreme susceptibility of skin to the deleterious effects of transplantation immunity reflects the presence of skin-specific histocompatibility antigens. Immunization of C3H/He mice with purified epidermal cells from H-2-compatible CBA mice leads to the generation of cytolytic T lymphocytes (CTLs) that recognize the epidermal-specific alloantigen Epa-1 in H-2-restricted fashion. In this report we describe four CTL lines that have been maintained in vitro for over 9 months that possess Epa-1 and H-2 restriction specificity identical to that observed in bulk CTL populations, as well as two CTL lines with novel specificities. The predominant cell surface phenotype of all of the CTL lines is Lyt-1-, Lyt-2+, and Thy-1+ as determined by indirect immunofluorescence. We could detect no evidence for overlap between these CTL lines and CTLs mediating lysis of target cells bearing foreign H-2 antigens. From these observations, we conclude that the CTLs in each of these long-term lines represent the progeny of a single clone whose specificity is directed toward tissue-specific alloantigens.