Non-heart-beating (NHB) donors are a valuable source of kidneys for transplantation. The organs, however, sustain substantial warm ischemic damage that may jeopardize the transplantability and result in nonfunction of the grafts. Quantification of warm ischemic time (WIT) and prediction of transplant outcome are essential for the use of NHB donor organs. During machine preservation (MP) the viability of NHB donor kidneys was evaluated through calculating intrarenal vascular resistance and determining lactate dehydrogenase and alpha-glutathione S-transferase (alphaGST) in the perfusate. Thirty-seven functioning (F) and nine nonfunctioning kidneys (NF) were compared. WIT was longer in NF; serum creatinine, donor age, and preservation time were not different. WIT correlated well with alphaGST after 4 and 8 hr of MP (r=0.353, P=0.009, and r=0.346, P=0.011, respectively). When compared with F, intrarenal vascular resistance was increased in NF after 4 and 8 hr of perfusion (P<0.05); at all time points, alphaGST levels were elevated in NF (P<0.05). Lactate dehydrogenase activity was not different between the groups, but could identify immediate functioning grafts within the F group. In conclusion, alphaGST levels correlated strongly with WIT and were also able to distinguish NF from F grafts. alphaGST can adequately predict the functional outcome of NHB donor grafts before transplantation; levels of alphaGST can be used to define reliable safety margins for viability. Therefore, MP is useful in evaluating the viability of NHB donor kidneys, and the parameters discussed will help to select nonviable grafts from this valuable pool of kidneys for transplantation.