The HERV-H family of endogenous retrovirus-like elements is widely distributed in the human genome, with about 1,000 full-length elements and a similar number of solitary long terminal repeats (LTRs). HERV-H LTRs have been shown to direct the transcription of both HERV-H-encoded and adjacent cellular genes. Transcripts of HERV-H elements are especially abundant in placenta, teratocarcinoma cell lines, and cell lines derived from testicular and lung tumors. Here we report that only a subset of HERV-H LTRs display promoter activity in human cell lines and that these LTRs are characterized by the presence of a GC/GT box immediately downstream of the TATA box. This GC/GT box is required for promoter activity, while, surprisingly, the TATA box is dispensable. The ubiquitously expressed transcription factors Sp1 and Sp3 bound to this GC/GT box and stimulated transcription from the promoter-active LTRs in the teratocarcinoma cell line NTera2-D1. However, in HeLa and Drosophila SL-2 cells, Sp1 acted as a transcriptional activator of the LTRs, while Sp3 acted as a repressor of Sp1-mediated transcriptional activation. Cotransfection studies also revealed that the tissue-specific Sp1-related protein BTEB bound to this GC/GT box and stimulated transcription from the LTR promoters in NTera2-D1 cells. These results show that members of the Sp1 protein family are crucial determinants for transcriptional activation of HERV-H LTR promoters and suggest that these proteins may also be involved in determining the tissue-specific expression pattern of HERV-H elements.