Second- to fourth-digit length ratio, 2D:4D, is a marker of testosterone level during foetal life that was found associated with sperm concentration or testosterone levels in some studies, but not in others, a difference possibly related to the way the ratio is assessed. In this study, 2D:4D was assessed in 122 men partners of pregnant women and in 71 testicular cancer patients using a new method based on direct measurements of finger lengths. In addition, we investigated the association between 2D:4D, birth weight, testicular volume, semen quality and time to pregnancy. A validation study of the method demonstrated high reliability and reproducibility. Neither digit lengths nor 2D:4D significantly differed in both groups of men. We found a significant negative association between 2D:4D and birth weight in testicular cancer patients. In fertile men, 2D:4D was associated with testicular volume (r=-0.36, p<0.001), total sperm number (r=-0.18, p=0.04) and time to pregnancy (r=0.24, p<0.02). In addition, participants with a history of epididymal cyst had a significantly higher 2D:4D than those without cysts. In conclusion, all significant findings indicate that the human male reproductive function is negatively related to 2D:4D. However, 2D:4D for testicular cancer patients does not point to a hormonal imbalance during foetal life as the common cause for developing germ-cell cancer. Such results obtained, thanks to an easy, direct and reliable method for measuring finger lengths, suggest the usefulness of this new tool in fertility studies as well as for studying men with developmental disorders of the reproductive tract.