In several cells and tissues the synthesis of HSP47, a collagen-specific molecular chaperone in the endoplasmic reticulum, is closely correlated with the synthesis of collagen. We previously reported that the Sp1 binding site at -210 bp in the promoter region and the first and second introns are required for the tissue-specific expression of HSP47 in transgenic mice (Hirata, H., Yamamura, I., Yasuda, K., Kobayashi, A., Tada, N., Suzuki, M., Hirayoshi, K., Hosokawa, N., and Nagata, K. (1999) J. Biol Chem. 274, 35703-35710). Here, we analyze how these introns influence the transcriptional regulation of the hsp47 gene in BALB/c 3T3 cells, which produce high levels of HSP47. In vitro promoter analysis using a luciferase reporter and gel mobility shift analysis revealed that two cis-acting elements in the first and second introns, BS5-B and EP7-D, respectively, are required for the activation of hsp47 in BALB/c 3T3 cells. Several members of the Kruppel-like factor (KLF) family of proteins were identified as BS5-B-binding proteins by yeast one-hybrid analysis using these elements as baits. One of these proteins, KLF-6/Zf9, binds to the BS5-B element and activates expression of the reporter construct when transfected into cells. Chromatin immunoprecipitation assay analysis revealed that the endogenous KLF-6/Zf9 binds the BS5-B elements that contain the CACCC motif, which is a consensus recognition sequence for other proteins in the KLF family. We also showed that BS5-B and EP7-D are bound by two members of the Sp1 family, Sp2 and Sp3. These results suggest that at least three sequences are required for the constitutive expression of hsp47 in BALB/c 3T3 cells: the -210 bp Sp1 binding site, the BS5-B element in the first intron, and the EP7-D element in the second intron. We suggest that KLF proteins regulate the transcription of hsp47 by binding the BS5-B element in cooperation with Sp2 and/or Sp3.