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Background: With a universal shortage of donor organs, screening and selection of marginal kidneys from non-heart-beating donors (NHBDs) provides a valuable source for transplantation. Pre-transplant viability assessment is based on a combination of flow characteristics and assessment of ischaemic tissue injury during NHBD kidney machine perfusion by measurement of total glutathione S-transferase (GST) activity. Successful viability screening has facilitated 69 renal transplants from 60 NHBDs within our transplant centre since 1998, with a first-year graft survival of 90.5%., Methods: We have investigated alanine aminopeptidase (Ala-AP) and fatty acid binding protein (FABP) as alternative biochemical markers to GST for pre-transplantation viability assessment. They were measured, together with GST, in tissue perfusate samples from machine-perfused kidneys from controlled and uncontrolled NHBDs., Results: During machine perfusion, a parallel response was seen for each of the three markers in the perfusates of controlled and uncontrolled NHBD kidneys over the 4-h perfusion. A highly significant correlation was obtained between GST and Ala-AP activities (P<0.0001) and between GST activity and FABP concentration (P<0.0001) in corresponding samples., Conclusion: In this study, GST, Ala-AP and FABP represent equivalent biochemical markers in terms of their ability to quantitate renal tissue injury in human NHBD kidneys. However, there may be some advantage in using all three analytes to provide complementary information on kidney allograft viability.