Microdissection testicular sperm extraction (TESE) has been used in the treatment of male infertility in cases of nonobstructive azoospermia (NOA). In some NOA cases, there is significant difficulty identifying sperm within the testes, and we propose that an immunofluorescence technique may assist in locating sperm foci for use in in vitro fertilization and intracytoplasmic sperm injection. The purpose of this study was to evaluate the feasibility of this novel technique. Thirteen fertile and 8 sterile mice were anesthetized, and a lower abdominal incision was performed to deliver the testes. Then a small incision was made into the tunica albuginea near the vascular pedicle to expose the seminiferous tubules. A microinjector fitted with a micropipette was used to inject fluorescein isothiocyanate-conjugated mouse anti-human acrosomal IgM antibody (HS-14) into adjacent injection sites along the exposed seminiferous tubules. Following antibody injection, the testes were examined under a Bio-Rad Multiphoton microscope to locate sperm through visualization of fluorescence. Sperm were identified in 22 of 26 testes from fertile mice either by morphologic appearance or by their motility, which was obvious under direct visualization. There was strong binding of the antibody to both the sperm head and tail. In contrast, no sperm were visualized in the sterile mice group. Use of an immunofluorescence technique during microdissection TESE for detection of sperm is a novel and feasible concept. Further studies of this technique are planned.