Plasma biochemical enzymes are commonly assayed as part of a diagnostic evaluation for zoological species, but their interpretation is complicated by a lack of knowledge about tissue of origin in many reptiles. This study evaluated tissue specificity of six biochemical enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase [ALP], creatine kinase [CK], gamma-glutamyl transferase [GGT], and glutamate dehydrogenase [GLDH]) in 10 tissues (skeletal muscle, cardiac muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, spleen, and reproductive tract) from 10 free-living eastern box turtles (Terrapene carolina carolina). CK activity was highest in skeletal muscle, cardiac muscle, and gastrointestinal tract; GLDH and ALT activities were highest in liver, kidney, and gallbladder; ALP and GGT activities were elevated in kidney and gastrointestinal tract; and AST was relatively nonspecific, with significantly higher activity in the cardiac muscle, liver, kidney, skeletal muscle, and gallbladder compared to other tissues (P < 0.05). These results serve as a first step toward improving clinical interpretation of plasma biochemistry panels in box turtles.