We evaluated brown and white adipose tissues for the presence of creatine, phosphocreatine and creatine phosphokinase activity. In rats 3.6 and 0.4 mumol of total creatine were found per g wet weight of brown and white adipose tissues, respectively. We were able to identify creatine by thin-layer chromatography after a pulse label of [14C]creatine had been given in vivo. Free creatine and phosphocreatine were shown to occur by column chromatography. Of the total creatine of brown adipose tissue, approximately one third to one half were attributable to phosphocreatine. The activity of creatine phosphokinase was demonstrated in both white and brown adipose tissue, the values of the latter prevailing over those of the former by a factor of 200, if based on wet weight, or 50, if expressed as specific enzyme activity. The labeling of total creatine in vivo proceeded much faster in adipose tissue than in skeletal muscle. The results strongly suggest that the energy metabolism of adipose tissue is closely dependent on the presence of creatine. The specific activities of free creatine and phosphocreatine of brown adipose tissue differed strikingly as long as 24 h after radioactive creatine was injected; this difference points to a metabolic or structural compartmentation of creatine.