The collagenous repeat-containing sequence of 26-kDa protein (CORS-26) was recently described as a new gene that is induced during adipocyte differentiation. Since the transcription factors specificity protein-1 (SP-1) and PPARgamma have been demonstrated to modulate transcriptional activation of adipocytic genes, we investigated the putative role of SP-1 and PPARgamma in the regulation of the murine CORS-26 promoter. Computer-based sequence analysis revealed two putative SP-1 binding sites and binding sites for PPARgamma and Pit-1 within the TATA-box containing promoter. Electrophoretic mobility shift assays (EMSA) with nuclear extracts from 3T3-L1 adipocytes and appropriate promoter fragments demonstrated that SP-1 binds specifically to both SP-1 binding sites. Specificity was demonstrated by (i) the appearance of supershift bands, (ii) competition experiments and, (iii) by using oligonucleotides carrying mutated SP-1 binding sites. Functional promoter activity was analyzed by Luciferase reporter gene assays and SP-1 was shown to exert inhibitory effects on the transcriptional activation of the murine CORS-26 gene. Additionally, specific binding activity of PPARgamma and Pit-1 to the CORS-26 promoter was demonstrated. Taken together, the present data demonstrate the functionality of the proximal murine CORS-26 promoter, which is regulated specifically by two SP-1 binding sites via SP-3-independent repressive effects of SP-1 on transcriptional activation. Pit-1 and PPARgamma can bind specifically to the promoter and might play an additive functional role in gene regulation of murine CORS-26.