The initial step of LINE1 retrotransposons dissemination requires transcription from species-specific promoter located within 5'-untranslated region of LINE1. Although the 5'-untranslated region of the rat LINE1 element shows promoter activity, no promoter-binding proteins have been discovered so far. Using an EMSA and Southwestern blotting methods, we identified Sp1 and Sp3 proteins, which specifically bind to the rat LINE1 promoter in vitro. The Sp1/Sp3-binding motif within rat LINE1 promoter is located downstream of the major predicted transcription initiation site. (c) 2005 Elsevier Inc. All rights reserved.