Intracytoplasmic sperm injection (ICSI) with testicular spermatozoa and ejaculated spermatids has been applied successfully to treat infertility because of azoospermia due to obstruction or testicular failure. In most cases, free spermatozoa are recovered from testicular tissue after mechanical mincing of multiple biopsies. Testicular sperm retrieval, however, remains unsuccessful in a high proportion of male patients suffering from testicular failure. In the present study, four media for enzymatic collagen digestion of testicular tissue were compared on 15 testis biopsies obtained from azoospermic patients following an ICSI treatment and on five biopsies from orchiectomy patients. To dissociate the testicular tissue, collagenase type IA, collagenase type IV, collagenase type IA supplemented with elastase and collagenase type IV supplemented with elastase were used. The preparations with only collagenase type IV provided the best dissolution of the cells from their tissue, with a higher yield of free vital testicular spermatozoa and single round germ cells. Enzymatic digestion of testicular tissue has the advantage, especially in cases of testicular failure, that several biopsies can be concentrated into one pellet of single cells. In this way, the sperm retrieval in cases of testicular failure might be optimized to yield the maximal numbers of spermatids and/or spermatozoa.