Abstract. The present study is part of a project on phospholipase D (PLD) in cardiac hypertrophy and analyzed effects on PLD activity of two growth stimuli, norepinephrine (NE) and cardiotrophin-1 (CT-1), in incubated rat heart. Phosphatidylcholine (PC) was labeled by 3 Hmyristic acid. PLD produced 3 H-phosphatidylethanol ( 3 H-PEth) from 3 H-PC in the presence of ethanol and maintained a basal formation of 3 H-PEth. Short-term and long-term exposure to NE for 2 or 13 h, respectively, enhanced the formation of 3 H-PEth, which was blocked by prazosin. Long-term pretreatment with NE or CT-1 increased the incorporation of 3 H-myristic acid into PC, which was blocked by atenolol. When the 3 H-PEth formation was expressed as a fraction of 3 H-PC, PLD activity seemingly was unchanged (NE) or markedly reduced (CT-1); the true effects, namely, stimulation by NE and nonresponsiveness towards CT-1, were unraveled by atenolol (NE) or when PLD activity was expressed as 3 H-PEth per ng protein.In conclusion, aadrenoceptor activation increased PLD activity. Long-term treatment with NE (via b-receptors) or CT-1 enhanced the 3 H-myristic acid incorporation into a PC compartment, that was not available for the a -receptor-mediated PLD activation. These results were discussed in regard to cellular mechanisms of cardiac hypertrophy and to the transphosphatidylation assay of PLD.