Short-term effects of 3,5-diiodo-L-thyronine (T₂) on lipid biosynthesis were studied in cultured hepatocytes from hypothyroid rats. A comparison with the effects of T₃ was routinely carried out. Following T₂ addition to cell cultures, a distinct stimulation of fatty acid and cholesterol syntheses, measured as incorporation of [1-¹⁴C]acetate into these lipid fractions, was observed. The T₂ dose-dependent effect on both metabolic pathways, already detectable at 10^-8-10^-9M, reached a 2-fold stimulation at 10^-5 M T₂. At this concentration, the stimulatory effect was evident within 1 h of T₂ addition to the hepatocytes and increased with time up to the length of the experimental period of 4 h. T₂ stimulation of lipogenesis was also confirmed by incubating hepatocytes with [³H]H₂O, used as an independent index of lipogenic activity. The effects of T₂ are rather specific as D-T₄ and 3,5-diiodo-L-tyrosine were practically ineffective on both fatty acid and cholesterol synthesis. Analysis of various lipid fractions showed that T₂ addition to the cells produced a significant stimulation of the incorporation of newly synthesized fatty acids into both neutral and polar lipids. By comparing the effects induced by T₂ with those seen in the presence of T₃, it appeared that T₂ was able to mimic T₃ effects. Experiments conducted in the presence of cycloheximide, a protein synthesis inhibitor, indicated that the T₂ stimulatory effect on fatty acid and cholesterol synthesis was essentially independent of protein synthesis.