To characterize the isoenzyme distribution of creatine kinase (CK) in endothelial cells (ECs) and its functional role during substrate depletion, ECs from aorta (AECs) and microvasculature (MVECs) of pig and rat were studied. In addition, high- energy phosphates were continuously monitored by ³¹P NMR spectroscopy in pig AECs attached to microcarrier beads. CK activity per milligram of protein in rat AECs and MVECs (0.08 ± 0.01 and 0.15 ± 0.08 U/mg, respectively) was <3% of that of cardiomyocytes (6.46 ± 1.02 U/mg). Rat and pig AECs and MVECs displayed cytosolic BB-CK, but no MM-CK. Gel electrophoresis of mitochondrial fractions of rat and pig ECs indicated the presence of mitochondrial Mi-CK, mostly in dimeric form. The presence of Mi_a-CK was demonstrated by indirect immunofluorescence staining using Mi_a-CK antibodies. When perifused with creatine-supplemented medium, phosphocreatine (PCr) continuously increased with time (1.2 ± 0.6 nmol · h¹ · mg protein¹), indicating creatine uptake and CK activity. Glucose withdrawal from the medium induced a rapid decrease in PCr, which was fully reversible on glucose addition, demonstrating temporal buffering of an energy deficit. Because both cytosolic and mitochondrial CK isoforms are present in ECs, the CK system may also contribute to energy transduction ("shuttle hypothesis").