Although protein tyrosine kinases (PTKs) signaling has been implicated in the late phase of ischemic preconditioning (PC), it is unknown whether PTK signaling is necessary for the development of nitric oxide (NO) donor-induced late PC. Thus, conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles followed by a 5-h recovery period of reperfusion for 3 consecutive days (days 1, 2, and 3). On day 0 (24 h before the 6 O/R cycles on day 1), rabbits received no treatment (control), the NO donor diethylenetriamine/NO (DETA/NO), the PTK inhibitor PP2 (PP2), or DETA/NO plus PP2 (DETA/NO+PP2). In control rabbits (n=6), the 6 O/R cycles on day 1 resulted in delayed functional recovery, indicating severe myocardial stunning. In rabbits pretreated with DETA/NO (n=5) on day 1, myocardial stunning caused by the 6 O/R cycles on day 1 was markedly attenuated, with a significant reduction (~60%) in the total deficit of WTh compared to controls, indicating that DETA/NO induced a late PC effect against stunning. However, in rabbits pretreated with DETA/NO plus PP2 (n=5), the total deficit of WTh was significantly greater than that in rabbits treated with DETA/NO alone and was similar to that in controls, indicating that PP2 prevented the development of DETA/NO-induced late PC. In rabbits pretreated with PP2 on day 0 (n=4), the total deficit of WTh was similar to that in controls, indicating that PP2 does not affect myocardial stunning in itself. We conclude that a PTK-dependent signaling mechanism is necessary for the development of NO donor-induced late PC against myocardial stunning in conscious rabbits.