BACKGROUND The extent of sperm DNA fragmentation, which can be measured by the TUNEL assay, is one of the determinants of male fertility. However, the clinical application of this test to in-vivo situations is difficult owing to the absence of a statistically validated threshold value. METHODS The aim of this study was to compare the results of TUNEL assay applied to semen samples from men of proven fertility (n = 47) and patients from an infertile population (n = 66), in order to establish a discriminating threshold value. RESULTS Infertile patients had a higher mean level of DNA fragmentation than men of proven fertility (40.9 +/- 14.3% versus 13.1 +/- 7.3%, respectively; P < 0.001). The area under the receiver operating characteristics curve was 0.93 for 20% sperm DNA fragmentation. The calculated threshold value for TUNEL assay to distinguish between fertile controls and infertile men was 20%. At this threshold, specificity was 89.4 [95% confidence interval (CI) 83.7-95.1] and sensitivity was 96.9% (95% CI 93.8-100). The positive and negative predictive values of the 20% sperm DNA fragmentation threshold were high: 92.8% (95% CI 87.9-97.5) and 95.5% (95% CI 91.6-99.3), respectively. CONCLUSION This study demonstrates that sperm DNA fragmentation, as measured by TUNEL assay, is a highly valuable indicator of male fertility.