Cultured keratinocyte allografts from unrelated donors can be readily grown as sheets in large-scale cell culture and have been used as an immediate skin cover for severely burned patients. Despite the absence of passenger leukocytes and the unlimited amount of material that can be obtained for permanent skin coverage, the allografts are susceptible to rejection. Since MHC class I (MHCI) antigens serve as targets for allograft rejection, we investigated whether 'phenotypic knockout' of human MHCI could be achieved through expression of an ER-directed anti-human MHCI single-chain intrabody (sFvhMHCI) that is directed against a monomorphic, conformational epitope, expressed across species lines, on the MHCI heavy chain. Co-immunoprecipitation of both MHCI heavy chain and beta2-microglobulin occurred in transfected monkey COS-1 cells, while Jurkat T cells stably expressing the ER-directed sFvhMHCI intrabody showed that complete phenotypic knockout of MHCI cell surface expression could be achieved. Infection of several human cell lines of divergent tissue sources and different HLA haplotypes resulted in marked down-regulation of MHCI expression, even under conditions where inflammatory cytokines (eg gamma-IFN) which up-regulate MHCI expression were used. Finally, when adenovirus encoding the anti-human MHCI intrabody was used to transduce primary human keratinocytes, a marked reduction of surface MHCI expression was observed. These in vitro studies set the groundwork for in vivo studies to determine if intrabody-mediated knockout of MHCI can impair alloantigen expression and prolong the survival of keratinocyte allografts.