Protein kinases regulate numerous cellular processes, including cell growth, metabolism, and cell death. Because the primary sequence and the three-dimensional structure of many kinases are highly similar, the development of selective inhibitors for only one kinase is challenging. Furthermore, many protein kinases are pleiotropic, mediating diverse and sometimes even opposing functions by phosphorylating multiple protein substrates. Here, we set out to develop an inhibitor of.a selective protein kinase phosphorylation of only one of its substrates. Focusing on the pleiotropic delta protein kinase C (delta PKC), we used a rational approach to identify a distal docking site on delta PKC for its substrate, pyruvate dehydrogenase kinase (PDK). We reasoned that an inhibitor of PDK's docking should selectively inhibit the phosphorylation of only PDK without affecting phosphorylation of the other delta PKC substrates. Our approach identified a selective inhibitor of PDK docking to delta PKC with an in vitro K-d of similar to 50 nM and reducing cardiac injury IC50 of similar to 5 nM. This inhibitor, which did not affect the phosphorylation of other SPKC substrates even at 1 mu M, demonstrated that PDK phosphorylation alone is critical for delta PKC-mediated injury by heart attack. The approach we describe is likely applicable for the identification of other substrate-specific kinase inhibitors.