A commercially available enzyme-immunometric assay for serum α-glutathione S-transferase (GST) was evaluated. Endogenous serum α-GST diluted linearly within the calibration range. However, we recommend that the sample and second antibody reagent are always added sequentially in the assay to avoid hook effect. Between-assay variability was below 7% across the calibration range and the upper limit of the reference range in adults (n = 219) was 11 · 4 μg/L. Within-individual variability in serum α-GST concentrations measured over a 4–6 week period in 4 healthy adults was small. Serum α-GST concentrations did not change significantly 6 h after a therapeutic dose of paracetamol. Studies in two patients after liver transplantation showed that serum α-GST is a better discriminant of acute changes in liver function than conventional tests. Serum α-GST concentrations were unaffected by gross muscle damage, extra-hepatic inflammation, or haemolysis and thus appear to be more liver specific than transaminase activities. The effect of renal impairment on serum α-GST concentrations requires further investigation.