The purpose of this study was to examine the time course of changes in plasma creatine kinase (CK), lactate dehydrogenase (LDH), aspartate (AST) and alanine aminotransferase (ALT) activity after intramuscular injection of 0.5% bupivacaine (BPVC). A total of 10 mL BPVC was injected into the biceps brachii (two sites, 5 mL per site) of five healthy, male subjects. Blood samples were obtained from the antecubital vein before and 2, 4, 8, 12, 24, 48, 72 and 96 h after the injection. Affected muscle size was visualized using magnetic resonance imaging (MRI), which was performed 4 days after the injection. Plasma CK activity started to increase 2 h and peaked 12 h after the injection. The peak CK activity (470 ± 62 IU L^-1 was approximately four times the pre-injection value (133 ± 24 IU L^-1), and no additional increase was observed alter 24 h. Plasma LDH, AST and ALT activities did not change significantly over time. Muscle around the injection sites showed increased T2 signal intensity using MRI. When smaller (2 mL or larger (20 mL) amounts of BPVC were injected into the biceps brachii in additional experiments, the amount of increase in plasma CK activity appeared to be related to the size of the affected muscle. It was concluded that CK started to leak from damaged muscle cells shortly after the BPVC injection, and the amount of increase in plasma CK activity appeared to reflect the amount of muscle damage.