The human genome contains more than half a million human endogenous retrovirus (HERV) long terminal repeats (LTRs) that can be regarded as mobile regulatory modules. Many of these HERV LTRs have been recruited during evolution as transcriptional control elements for cellular gene expression. We have cloned LTR sequences from two HERV families, HERV-H and HERV-L, differing widely in their activity and tissue specificity into a murine leukemia virus (MLV)-based promoter conversion vector (ProCon). Various human cell lines were infected with the HERV-MLV hybrid vectors, and cell type-specific expression of the reporter gene was compared with the promoter specificity of the corresponding HERV LTRs in transient-transfection assays. Transcription start site analysis of HERV-MLV hybrid vectors revealed preferential use of the HERV promoter initiation site. Our data show that HERV LTRs function in the context of retroviral vectors in certain cell types and have the potential to be useful as cell type-specific promoters in vector construction.