We investigated the effects of the leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) on 3,3', 5-triiodo-L-thyronine, or thyroid hormone (T(3))-stimulated sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) gene expression on cultured neonatal rat cardiac myocytes. A reduction of T(3) induced increases in SERCA2 mRNA levels after co-treatment with LIF or IL-6. To investigate for the molecular mechanism(s) responsible for the blunted gene expression, a 3.2-kb SERCA2 promoter construct containing a reporter gene was transfected into cardiac myocytes. T(3) treatment stimulated transcriptional activity twofold, whereas co-treatment with T(3) and either of the cytokines caused an inhibition of T(3)-induced SERCA2 transcriptional activity. A T(3)-responsive 0.6-kb SERCA2 construct also showed a similar inhibition by cytokines. Cytokine inhibition of SERCA2 transcriptional activity was also evident when a 0.6-kb SERCA2 mutant, T(3)-unresponsive promoter construct was used. Treatment with T(3) and cytokines showed a significant decrease in transcription when a reporter construct was used that was comprised of direct repeats of SERCA2 thyroid response element I. These data provide evidence for cytokine-mediated inhibitory effects on the SERCA2 promoter that may be mediated by interfering with T(3) action.