The mitochondrial uncoupling protein gene is rapidly induced in mouse brown fat following cold exposure. To identify cis-regulatory elements, approximately 50 kb of chromatin surrounding the uncoupling protein gene was examined for its hypersensitivity to DNase I. Seven DNase I-hypersensitive sites were identified in the 5'-flanking DNA, and one site was identified in the 3'-flanking DNA. Transgenic mice with an uncoupling protein minigene were generated by microinjection of fertilized eggs with a transgene containing 3 kb of 5'-flanking DNA and 0.3 kb of 3'-flanking DNA. Expression of the transgene is restricted to brown fat and is cold inducible. Four additional transgenic lines were generated with a second transgene containing a 1.8-kb deletion in the 5'-flanking DNA, and expression of this minigene is absent in all tissues analyzed. A DNase I-hypersensitive site located in the 1.8-kb deletion contains a cyclic AMP response element that binds a brown fat tumor enriched nuclear factor. On the basis of these observations, we propose that a cis-acting regulatory sequence between -3 and -1.2 kb of the 5'-flanking region, possibly at a DNase I-hypersensitive site, is required for controlling uncoupling protein expression in vivo.