Triiodo-L-thyronine (T3) added in vitro to fat pads from normal, or propylthiouracil-treated rats enhanced the rate of release of glycerol and free fatty acids (FFA) in the presence of epinephrine. An effect of T3 was also demonstrated in the presence of adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone, or glucagon in studies with tissue from normal rats. The minimal effective concentration of T3 was approximately 2.5 × 10-5 mole/liter for intact fat pads and 3 × 10-6 mole/liter for fat cells. With fat pads from propylthiouracil-treated rats the effect of T3 was not apparent until the 3rd hr of incubation. Enhancement of epinephrine-stimulated lipolysis by T3 was evident during the 1st hr of incubation of fat pads from normal rats, and fat cells responded almost immediately to the presence of T3. When added alone or in the presence of theophylline, 3′,5′-adenosine monophosphate or its dibutyryl derivative, T3 had little or no effect on lipolysis. The effect of T3 was observed with or without glucose in the medium, and was not inhibited by cycloheximide or actinomycin D. It did not persist when tissues, after incubation in the presence of T3 were transferred to medium without T3. No effect of T3 on glucose uptake in the presence of epinephrine, ACTH, or insulin was demonstrated.