The effect of thyroid hormones on fat mobilization induced by epinephrine in adipose tissues of fasted rats was studied both in vivo and in vitro. Thyroidectomy markedly diminished, but did not completely abolish, the lipolytic response to adipose tissue to epinephrine. Triiodo-L-thyronine (L-T3) added to a suspension of fat cells at a concentration of as low as 0.01 mM stimulated epinephrine-induced lipolysis. Even in the absence of epinephrine, L-T3 at concentrations above 0.1 mM clearly stimulated the basal lipolysis of fat cells from both thyroidectomized and euthyroid rats. Various compounds structurally related to L-T3 were tested, but only diiodo-L-thyronine (L-T2) showed a similar stimulatory effect on lipolysis. Adenosine 3′, 5′-monophosphate (cyclic AMP) could not replace epinephrine in stimulating lipolysis, but increased the release of free fatty acid from fat cells in the presence of L-T3. Theophylline stimulated lipolysis, and its effect was enhanced by L-T3. At a high concentration of theophylline (10mM), the lipolytic response of fat cells was almost maximal regardless of the absence or presence of L-T3, with or without epinephrine. Furthermore, both L-T3and L-T2 were found to inhibit cyclic AMP phospho-diesterase effectively, indicating that thyroid hormones might elicit a lipolytic action by preventing destruction of cyclic AMP.