Mitochondrial acyl CoA:glycine N-acyl transferase (ACGNAT) was purified to homogeneity from adult human liver. It was found to be a monomer of 30 kD, having a pI of 6.8. ACGNAT retained 47% of enzymatic activity at 100 mM NaCl concentration, whereas 21% of the activity was retained with KCl and 32% with K3PO4 at 100 mM concentration as compared to the control. The stability studies revealed no change in activity at 4 degrees C for up to 72 h, 25 degrees C for 4 h and at 37 degrees C for 1 h. The Km values of human ACGNAT for benzoyl CoA, salicyl CoA, isovaleryl CoA and octanoyl CoA were 57.9, 83.7, 124 and 198 mM, respectively, and the corresponding Vmax values were 17.1, 10.1, 7.64 and 3.3 mumol/min/mg protein. The availability of pure human ACGNAT would help in studying the molecular genetics and structural biology of this protein which is important in the detoxification of various endogenous and xenobiotic acyl CoA's.