Primary cultures of brown adipocytes were used to investigate the regulation of malic enzyme (ME) gene expression by insulin and T3. No ME gene expression was detected in undifferentiated preadipocytes. The levels of ME mRNA increased slightly during cell differentiation. Physiological doses of insulin or T3 increased ME gene expression, which reached a maximum after 24 h, on whichever culture day they were added. The effects of insulin and T3 were at the transcriptional level, as measured by run-on assays. Both hormones also increased the stabilization of the transcripts and required ongoing protein synthesis to exert their effects. A comparison of the potencies of insulin and insulin-like growth factor-I and -II (IGF-I and -II) in this system indicated that induction by insulin is mediated via its own receptor. The effects of insulin and T3 were independent of the extracellular glucose concentration, but were additive to that of glucose. Moreover, insulin and T3 act additively to increase ME gene expression, suggesting that they interact either at the transcription level or that of mRNA stabilization.