Recovery of testicular spermatozoa from azoospermic patients with testicular failure, followed by intracytoplasmic sperm injection (ICSI) is a recent advance in the treatment of male infertility. In most cases, free spermatozoa are recovered from testicular tissue after mechanical mincing of multiple biopsies. Testicular sperm retrieval, however, remains unsuccessful in 30-50% of male patients suffering from Sertoli cell-only syndrome and maturation arrest. In this study, a strategy was developed in order to maximize the chance of sperm retrieval in difficult cases of testicular failure. The ultimate step was the use of enzymatic procedures (collagenase type IV) to dissociate the testicular tissue completely. Testicular tissue of 41 patients for whom no spermatozoa were found after mechanical mincing of the testicular tissue was investigated. In 14 out of the 41 cases (34%), enough spermatozoa for ICSI were found after fine mincing of multiple biopsies and several hours' search in the cell suspension treated with the erythrocyte-lysing buffer (ELB). In 27 out of the 41 patients, no spermatozoa were found even after the use of ELB. In seven out of these 27 failures (26%), spermatozoa for ICSI were retrieved after enzymatic dissociation of the residual minced tissue pieces, thus making ICSI possible despite failure to find spermatozoa with conventional mincing. From this study, we may conclude that enzymatic digestion of testicular tissue is easy to perform, is not time-consuming and constitutes a successful method in reducing the sperm recovery failures in patients with non-obstructive azoospermia.