Vascular complications associated with diabetes mellitus (DM) have been linked to activation of protein kinase C (PKC)-dependent signaling pathways in both human and animal models of DM. To determine whether aberrant PKC signaling mechanisms specifically impact the coronary circulation, we assessed isolated coronary artery (CA) responses following induction of Type I DM. Male Sprague-Dawley rats were subjected to partial pancreatectomy (DM; n=23) and compared to age-matched controls (CTL; n=19). Vasoreactivity was assessed in single CAs (~250 µm ID) following abluminal administration of the G_q-dependent vasoconstrictors Endothelin-1 (ET-1; 10^-10-10^-9 M) and U44619 (10^-9-10^-5), or the voltage-gated Ca²⁺ channel agonist, Bay K 8644 (10^-9-10^-5), with and without the PKC inhibitor bisindolylmaleimide (Bis; 10^-6 M). Dilator responses to Acetylcholine (Ach; 10^-9-10^-5 M) were also assessed. ET-1 resulted in significantly greater constriction in DM vs. CTL (50 ± 4% vs. 33 ± 5%; P <0.0001), while responses to U44619 and Bay K 8644 were similar between groups. Importantly, inhibition of ET-1 and U44619 constriction by Bis occurred in DM but not CTL (P<0.05). Western blotting on isolated CA revealed greater levels of PKC, PKCI and PKCII by 22%, 15.3% and 17.6%, respectively in DM vs. CTL (P<0.05), while PKC and PKC protein levels were unchanged. DM was also associated with attenuated CA dilation following Ach (P<0.0566) and reductions in endothelial nitric oxide synthase (eNOS) protein levels vs. CTL (P<0.03). These data suggest that Ca²⁺-dependent PKC signaling pathways, particularly for ET-1, play a greater role in modulating CA vasoconstrictor responses in DM vs. CTL. These data further suggest that aberrant CA constrictor and dilator responses are likely to contribute to coronary vascular pathology associated with DM.