Objective: Intracytoplasmic sperm injection (ICSI) of testicular sperm is most efficacious when the sperm selected for injection is alive. Procurement of motile sperm is a clear means of ensuring vitality. In this study, testicular biopsies were subjected to motility enhancement either by testicular biopsy culture prior to cryopreservation, by exposure to chemical stimulants post-thaw, or by the two combined. The results of a trial thaw were used to guide sperm preparation for assisted reproduction. Design: This case management study was performed with 7 couples in which the man was azoospermic because of obstruction. Testis biopsies were divided prior to freezing into cultured and non-cultured groups and further divided after thaw into media with or without a chemical stimulant. The four resulting groups were compared in yield of progressively motile sperm. The optimal procedure was used in subsequent attempts to fertilize the partner’s ova by ICSI. Materials/Methods: Testis biopsy from beneath the tunica albuginea was dissected into small fragments and frozen by a standard method immediately after surgery or after 48-72 hours of in vitro culture. For the trial, cultured and non-cultured specimens were thawed, divided again and washed in the presence or absence of 2 mM 2-deoxyadenosine + 3.5 mM pentoxifylline (later pentoxifylline alone). Each suspension was concentrated to 50 microliters, placed under oil, and evaluated for progressively motile sperm. On the day of egg retrieval, sperm were prepared by the least invasive method that had produced sufficient motile sperm in the trial. When stimulant was used, sperm were washed exhaustively in a stimulant-free drop of PVP prior to injection. Clinical outcomes were evaluated. Results: Progressively motile testicular sperm were rare in thawed specimens in the absence of biopsy culture. Cultured testicular specimens displayed motile sperm at the edge of the microdrop in 6/6 trial thaws without stimulant. However, progressively motile sperm increased 2.7-fold with stimulant. Thirteen attempts to conceive by ICSI were performed using thawed testicular sperm. The overall rates of 2pn formation, implantation, and clinical pregnancy were 74%, 24%, and 62%. The corresponding rates for ejaculated sperm in our program were 77%, 20% and 41%. Two of 13 attempts were made with sperm from biopsy culture alone and resulted in 2 pregnancies and 3 normal babies. One attempt performed with sperm from non-cultured biopsy exposed to stimulant resulted in a singleton live birth. Five of 10 attempts with sperm from biopsy culture and motility stimulant in combination resulted in clinical pregnancy. Of these, four patients delivered 6 normal offspring and one resulted in early loss. Conclusions: We confirmed reports that in vitro culture of testicular tissue results in stimulation of progressive sperm motility. In only 2 of 13 cases, however, were we confident that this alone would provide enough motile sperm for ICSI after a freeze-thaw. Post-thaw treatment of non-cultured or cultured testicular sperm with stimulant resulted in a significant enhancement of motility and allowed predictable availability of motile sperm for subsequent ICSI. Microinjection of ova with testicular sperm subjected to biopsy culture and/or motility stimulant treatment resulted in the birth of 10 normal infants and one first trimester loss. Supported By: Local funds.