The beta(3)-adrenergic receptor (beta(3)AR) is expressed predominantly in adipocytes, and it plays a major role in regulating lipolysis and adaptive thermogenesis. Its expression in a variety of adipocyte cell models is preceded by the appearance of CCAAT/enhancer-binding protein alpha (C/EBP alpha), which has been shown to regulate a number of other adipocyte-specific genes. Importantly, it has been demonstrated that several adipocyte cell lines that fail to express C/EBP alpha exhibit reduced insulin sensitivity, despite an apparent adipogenic phenotype. Here we show that transcription and function of the beta(3)AR correlates with C/EBP alpha expression in these adipocyte models. A 5.13-kilobase pair fragment of the mouse beta(3)AR promoter was isolated and sequenced. This fragment conferred a 50-fold increase in luciferase reporter gene expression in adipocytes. Two putative C/EBP binding sites exist at -3306 to -3298 and at -1462 to -1454, but only the more distal site is functional. Oligonucleotides corresponding to both the wild-type and mutated -3306 element were inserted upstream of a thymidine kinase luciferase construct. When cotransfected in fibroblasts with a C/EBP alpha expression vector, reporter gene expression increased 3-fold only in the wild-type constructs. The same mutation, when placed into the intact 5.13-kilobase pair promoter, reduced promoter activity in adipocytes from 50-fold to <10-fold. Electrophoretic mobility shift analysis demonstrated that the site at -3306 generated a specific protein-oligonucleotide complex that was supershifted by C/EBP alpha antibody, while a probe corresponding to a putative site at -1462 did not. These results define C/EBP alpha as a key transcriptional regulator of the mouse beta(3)AR gene during adipogenesis.