Synthesis of phosphatidylcholine (PC) by S-adenosylmethionine-dependent methylation of phosphatidylethanolamine has previously been associated with receptor-mediated histamine and pituitary hormone secretion. We investigated stimulation of phospholipid methylation by TSH and its possible role in thyroid hormone secretion. Rat hemithyroids were incubated in Krebs-Henseleit-glucose-BSA buffer and the effect of various treatments on the incorporation of [3H-methyl]L-methionine into PC and T4/T3 secretion was studied. TSH treatment elevated thyroid phosphatidylethanolamine methyltransferase activity, the incorporation of [3H-methyl]methionine into PC, and T4/T3 secretion. The increase in PC synthesis was linear up to 6 h in a dose-dependent fashion (half-maximal stimulation at 2.5 micrograms TSH/ml). Stimulation required protein synthesis, because cycloheximide inhibited the increase in PC synthesis by 77%. Inhibitors of phospholipid methylation (100 microM adenosine + 10 microM L-homocysteine thiolactone + 10 microM erythro-9[2-hydroxy-3-nonyl]adenine) significantly decreased TSH-stimulation of phospholipid methylation but not T3/T4 secretion. In conclusion, stimulation of thyroid phospholipid methylation by TSH is not required for stimulated secretion of thyroid hormones.