The transglutaminase I (TGase I) gene encodes an enzyme that catalyzes the cross-linking of structural proteins involved in the formation of the cornified envelope during squamous cell differentiation. To identify DNA elements important for the transcriptional control of the TGase I gene, we analyzed the ability of a 2.9-kilobase pair (kb) upstream regulatory region to control the expression of a reporter gene in vivo and in vitro. Transgenic mice bearing the pTG(-2.9kb)CAT construct exhibited the same pattern of tissue-specific expression of CAT as reported for TGase I. Deletion analysis in transiently transfected rabbit tracheal epithelial cells indicated that two sequences from bp -490 to -470 and from -54 to -37 are involved in the activation of TGase I transcription. Point mutation analysis and mobility shift assays showed that the sequence located between -54 and -37 is a functional Sp1-like transcription element. Sp1 and Sp3, but not Sp2, are part of nuclear protein complexes from differentiated RbTE cells binding to this site. The element TGATGTCA between bp -490 and -470 is contained in a larger 22-bp palindrome and resembles the consensus cAMP response element-binding protein (CREB)/AP-1 element recognized by dimeric complexes of members of the CREB, ATF, Fos, and Jun families. Mutations in this sequence greatly reduced promoter activity. Supershift analysis identified CREB1, JunB, c-Fos, Fra-1, and c-Jun in protein complexes isolated from differentiated rabbit tracheal epithelial cells binding to this site. Our study shows that the Sp1- and CREB/AP-1-like sites act in concert to stimulate transcription of the TGase I gene. The 2.9-kb promoter region could guide expression of specific genes in the granular layer of the epidermis and could be useful in gene therapy.