Previous research has demonstrated that a transient increase in interstitial adenosine and subsequent activation of ATP-sensitive K+ (KATP) channels are involved in triggering ischemic preconditioning (PC), however, the role of adenosine in mediating the cardioprotection of hypoxic PC and that produced by KATP channel openers is less clear. Thus, the aim of the present study was to determine the role of adenosine in mediating the cardioprotective effects of PC produced by 5 min of ischemia, hypoxia, or by a 5-min intracoronary (i.c.) infusion of the KATP channel opener bimakalim (1 microgram/min). A single microdialysis probe was implanted into the midwall of the ischemic area for sampling of interstitial fluid adenosine and its breakdown products during the PC stimulus, prolonged occlusion (60 min) and during the first 30 min of the reperfusion (3 h) period. Ischemic, hypoxic and bimakalim pretreatment significantly reduced infarct size, 5.3 +/- 1.5; 8.9 +/- 2.5; 11.4 +/- 3.2, respectively, as compared to control: 27.3 +/- 6.5. Both ischemic and hypoxic PC produced similar and significant increases (0.56 +/- 0.13 mumol/l to 1.12 +/- 0.12 mumol/l and 1.32 mumol, control, ischemic and hypoxic PC, respectively) in dialysate adenosine concentration which persisted during the brief 10-min reperfusion period following PC. However, i.c. bimakalim resulted in a significant decrease in dialysate adenosine (0.56 +/- 0.13 mumol/l to 0.22 +/- 0.04 mumol/l) which persisted during the 10-min drug-free period. All three PC protocols resulted in similar decreases in dialysate adenosine, inosine and uric acid concentrations throughout the prolonged ischemic period as compared to control animals. In conclusion (1): PC produced by ischemia or hypoxia results in an increase in interstitial adenosine prior to a prolonged occlusion period; (2) the KATP channel agonist, bimakalim, significantly decreased interstitial adenosine prior to a prolonged occlusion period; (3) ischemic PC, hypoxic PC, and bimakalim pretreatment produced a similar reduction in interstitial adenosine and its breakdown products during the prolonged ischemic period. These results suggest that an increase in interstitial adenosine may be necessary for the initiation of the protective effect of ischemic and hypoxic PC but an increase in adenosine is not necessary for the cardioprotective effect of a direct opener of the KATP channel.