Iodothyronine 5'-deiodinase (5'D) of mouse brown adipocytes differentiated in cell culture was characterized in detail with respect to the adrenergic control of its biosynthesis. The stimulation of 5'D required mRNA and protein synthesis and was dependent on the stage of differentiation of the cells. The maximum induction was observed around confluence (7-day-old cells), in pre- and post-confluent cells the 5'D activity was significantly less induced. The transient responsiveness of brown fat-cells to the stimulatory effect of adrenergic agents was reflected also in the time course of the induction of 5'D by different concentrations of agonists. The maximum response occurred regularly after an 8 h incubation and implicated a rather fast turnover of the induced enzyme. On the basis of the inhibitory effects of cycloheximide and actinomycin D, the half-life of the induced 5'D and its mRNA were estimated to be 1.5 and 3.3 h respectively. The noradrenaline-induced 5'D activity was shown to be that of the type II enzyme, insensitive to propylthiouracil (PTU). The estimated values of its apparent Km for thyroxine, Km for the co-substrate dithiothreitol, and Vmax. in the presence of 1 mM PTU were 2 nM, 2.6 mM, and 0.1 pmol of I-/h per mg of protein respectively. The 5'D activity was effectively induced by forskolin and dibutyryl cyclic AMP, as well as by isoprenaline, noradrenaline and CGP-12177, but not by phenylephrine, cirazoline or oxymetazoline. This indicates that, contrary to previous observations in vivo, stimulation of 5'D in cultured brown fat-cells involves elevated cyclic AMP levels and is mediated predominantly via beta-receptors, particularly via the so-called beta 3-adrenoceptors.