CCAAT/enhancer binding proteins (C/EBP) are a family of transcription factors with a highly conserved basic/leucine zipper (bZIP) domain that has been implicated in the transcriptional control of genes involved in cell growth and differentiation. We have previously demonstrated that the expression of C/EBPalpha and C/EBPbeta genes is regulated by thyroid hormone in rat liver during development. The aim of the present study was to explore the molecular mechanisms underlying the control of C/EBPalpha gene expression by thyroid hormone. To achieve this goal, we isolated and characterized a genomic clone containing 1171 bp of the 5'-flanking region of the rat C/EBPalpha gene. This fragment was an active promoter in MB492 cells, an immortalized brown adipocyte cell line that expresses the endogenous C/EBPalpha gene in a T3-dependent manner. Sequence analysis suggested the presence of three thyroid hormone response elements, TRE-1 (-602/-589), TRE2 (-411/-396), and TRE3 (-376/-350). The results of deletion, mutagenesis, and gel mobility shift analysis disclosed that only TRE-1, an ER2-type response element, represented a functional T3 response element. Our results demonstrate that T3 is a factor that positively regulates C/EBPalpha gene expression in a direct fashion.