Examination of the in vivo effect of 2,4-dinitrophenol (DNP) on the brown adipose tissue of cold-exposed rats, as well as the in vitro response of this tissue to DNP and dicumarol, indicates that brown fat does possess a functional electron transport coupled phosphorylating system. Moreover, the fact that a norepinephrine-induced thermogenic response (in vivo) can be elicited from the brown fat after DNP administration implies that the effect of norepinephrine (NE) is not primarily due either to a physiological uncoupling by fatty acids, the level of which is increased by NE, or to stimulation of an ATP-ase system. Alternatively, our data suggest that under basal conditions (i.e. when the animal is not stimulated by cold stress or NE), the heat production (oxygen consumption) of the brown fat is limited by the availability of substrate rather than ADP. It is thus proposed that the thermogenic effect of NE results from the stimulation of lipolysis and an attendant increase of substrate available for oxidation.