The long terminal repeat (LTR) sequences of Moloney murine leukemia virus and its closely related derivative Moloney murine sarcoma virus (Mo-MSV) are incapable of directing transcription in embryonal carcinoma (EC) stem cells. The myeloproliferative sarcoma virus, a derivative of Mo-MSV, has several point mutations in the LTR and is transcribed more efficiently to allow productive infection of F9 EC cells. One of these mutations, at -166 with respect to the transcriptional start, creates a consensus binding site for the well-characterized mammalian transcription factor Sp1. We used gel retardation assays to demonstrate that F9 EC cell extracts form several complexes with the myeloproliferative sarcoma virus sequence around -166. One of these complexes involves a murine Sp1-like protein, which has immunoreactivity, DNA binding specificity, and electrophoretic mobility equivalent to those of purified human Sp1 protein. An equivalent complex forms on the corresponding Mo-MSV sequence but with a fivefold-lower affinity. Consistent with these observations, introduction of the single point mutation at -166 into the Mo-MSV LTR, creating a consensus Sp1 binding site, increases expression in F9 EC cells sixfold.