We used three interventions to test critically the theory that ischemic preconditioning is the result of translocation of cytosolic protein kinase C (PKC) into the membranes where it can be activated. If that theory were true then kinase activity should not be necessary during the preconditioning ischemia and thus blocking kinase activity at this time should not block protection. Secondly, since most translocation processes in the cell are accomplished by cytoskeletal microtubules, disrupting them with colchicine should also block protection from preconditioning. Finally, translocating PKC by transient exposure to PMA, should still require adenosine receptor activation to reactivate the PKC pathway during the subsequent ischemia. Blocking kinase activity with staurosporine during a 30 min insult completely blocks protection in preconditioned hearts but when staurosporine treatment was confined to the preconditioning episode protection was not blocked in five of the eight hearts studied. Microtubule disruption with colchincine did block the protective effect of preconditioning (38.3 +/- 1.9% infarction v 40.6 +/- 4.1% in non-preconditioned). Colchicine had no effect on infarct size in the non-preconditioned group. Five min PMA treatment plus 10 min washout significantly limited infarct size in isolated rabbit hearts subjected to 30 min regional ischemia (5.9 +/- 1.1% v 31 +/- 3.5% infarction in control). PMA's protection was blocked by adding the adenosine receptor blocker, SPT, during the sustained ischemia (38.1 +/- 6.1% infarction). All three of these experiments strongly support the translocation theory of ischemic preconditioning.