BACKGROUND AND PURPOSE Nicotinamide adenine dinucleotide (NADH) diaphorase staining has been used to confirm cell viability or death after radiofrequency ablation (RFA) of renal tissue. The time course over which NADH staining status converts from viable to non-viable after a lethal insult has not been defined for renal RFA, but the change may not be immediate. Our objective was to assess porcine renal tissue for viability using NADH diaphorase staining at various times after RFA. MATERIALS AND METHODS Seven pigs underwent monopolar RFA of both kidneys followed by needle biopsy of the ablation zone before and immediately after ablation and at 15-minute intervals thereafter. Initially, a single kidney was treated, and the contralateral kidney was treated 2 weeks later. Biopsies were taken from untreated renal parenchyma in a similar time course after nephrectomy to examine the effect of ischemia. All biopsy specimens, as well as representative sections of the ablation zone, were subjected to NADH staining and reviewed by a pathologist who was blinded to the tissue treatment. RESULTS Most of the post-RFA biopsy specimens (86%) showed non-viable tissue. However, 14% of the specimens revealed viable tissue as late as 150 minutes after RFA. Therefore, none were positive. In the nephrectomy parenchyma, 92% of the biopsy specimens showed viable tissue as late as 4 hours after the onset of ischemia. CONCLUSION Staining for NADH can establish tissue non-viability after RFA, but the timing of staining after treatment must be considered when interpreting results to avoid false positive tests. Tissue that is apparently viable by NADH staining within 2.5 hours of RFA may in fact have been ablated.