Objective: To study the effect of in vitro culture on the quality of human testicular sperm and the efficiency of intracytoplasmic sperm injection with in vitro cultured testicular sperm. Design: Clinical study. Setting: A private IVF center. Patient(s): Twenty consecutively seen IVF patients undergoing testicular biopsies for ICSI. Intervention(s): The testicular specimens were cultured in vitro for 24 hours and the isolated spermatozoa were microinjected. Main Outcome Measure(s): Preincubation and postculture sperm motility, and fertilization, implantation, and pregnancy rates after intracytoplasmic sperm injection. Result(s): Motility increased from initial nonmotile or twitching sperm to free motile sperm in 18 of 20 cases. The injection of in vitro cultured testicular sperm resulted in a fertilization rate of 58%, an implantation rate of 20%, and a pregnancy rate of 45%. Conclusion(s): A testicular biopsy procedure can be performed the day before egg retrieval. Despite the low initial sperm quality, a high percentage of the prepared testicular sperm showed increased motility after 24 hours of culture. The injection of in vitro cultured testicular sperm into matured oocytes resulted in fertilization, implantation, and pregnancy rates comparable to those obtained with ejaculated sperm.