Activation of protein kinase C (PKC) by the phorbol ester phorbol 12-myristate 13-acetate (PMA) has been shown to shorten the time to turn on ATP-sensitive potassium currents (I_K,ATP) during metabolic inhibition (MI) but only when adenosine (Ado) is included. In the present study we tested whether pretreatment with Ado could mimic the effect of PMA in isolated rabbit ventricular myocytes. When I_K,ATP was measured by conventional whole cell clamp, pretreatment with 100 µM Ado did not alter the time to I_K,ATP activation: 13.5 ± 2.1 vs. 12.4 ± 1.9 min with Ado during MI. We repeated the experiment using the perforated patch technique. Consistent with the previous results in conventional whole cell patch recordings, the time to turn on I_K,ATP during MI (with Ado included) was shortened from 27.1 ± 2.2 to 12.6 ± 2.4 min (P < 0.01) when cells were pretreated with PMA and Ado was included during MI. In contrast to conventional whole cell recordings, Ado pretreatment also abbreviated the time for I_K,ATP activation during MI (with Ado included) to 16.4 ± 1.8 min. This effect was partially eliminated by simultaneous administration of an Ado receptor blocker or a PKC inhibitor during Ado pretreatment, suggesting that pretreatment with Ado stimulates PKC by activating Ado receptors. Our results demonstrate that Ado can prime I_K,ATP during subsequent MI in the presence of Ado. This priming effect appears to be mediated by PKC upregulation of the channel. These results support the notion that Ado plays a dual role to initiate and to mediate ischemic preconditioning and links the roles of Ado receptors, PKC, and I_K,ATP in ischemic preconditioning.