Endothelin-1 (ET-1) is believed to play an important role in cardiac ischaemia/reperfusion injury. ET-1 is synthesized from preproET-1 by the action of ET-converting enzyme (ECE). It is unclear to what extent the ET system is activated following prolonged ischaemia. In this study we used a model mimicking the conditions of the donor heart during transplantation. Isolated rat hearts perfused with Krebs-Henseleit buffer were subjected to 30 min of normothermic perfusion, then 4 h of cardioplegic arrest at 4 degrees C with St Thomas' Hospital solution, followed by reperfusion for 2 h. Hearts were freeze-clamped at different time points during the protocol. Using quantitative reverse transcription-PCR, relative levels of ET-1 and ECE mRNA expression were measured and compared with a housekeeping gene (ribosomal protein L32). During reperfusion there was a consistent decrease in coronary flow to approx. 85-90% of pre-ischaemic flow. There was no significant alteration in preproET-1 mRNA expression during 2 h of reperfusion. However, ECE mRNA expression was increased by 77.5% at 1 h and by 74.6% at 2 h following ischaemia compared with pre-ischaemic values (P<0.05). Thus we conclude that ECE mRNA expression is increased following prolonged hypothermic cardioplegic arrest. Elevations in the expression of this enzyme may help to explain the role of the ET system in the pathogenesis of ischaemia/reperfusion injury following cardiac surgery and transplantation.