Expression of the gene for the brown-fat specific uncoupling protein thermogenin was investigated in cell cultures by hybridization of isolated RNA with a cDNA clone corresponding to mouse thermogenin. The RNA was isolated 3-4 days after confluence from cells differentiated in culture from precursors isolated from the interscapular brown adipose tissue of 5-week-old mice. Very low thermogenin mRNA levels were found in cells derived from untreated mice, and there was only little effect of added norepinephrine on thermogenin gene expression in these cells. However, in cells derived from hypothyroid (methimazole-treated) mice there was a higher expression of thermogenin, and norepinephrine had a marked augmenting effect on the thermogenin mRNA level in these cells. These effects of thermogenin mRNA levels were specific, in that they contrasted with the effects of hypothyroidism and norepinephrine on the level of other mRNA species in these cells (coding for beta-actin, lipoprotein lipase, cytochrome-c oxidase, and glycerol-3-phosphate dehydrogenase). It was concluded that brown-fat cells in culture can reach a differentiated state, sufficiently advanced that the unique properties of these cells can be expressed, and that thermogenin gene expression (i.e., the level of thermogenin mRNA) is under direct control of norepinephrine.