We evaluated the potential use of a non-contact, 1.48 microm wavelength diode laser for immobilization of human spermatozoa and permeabilization of the sperm membrane in different culture media. When we applied a single laser shot near to the middle region of the sperm tail, spermatozoa could be immobilized either temporarily or permanently, depending on the energy used. Above an energy of 2 mJ in polyvinylpyrrolidone and 2-3 mJ in culture medium, a reliable permanent immobilization was achieved by permeabilization of the sperm tail membrane. We then explored the use of a double laser shot technique. Spermatozoa were temporarily immobilized by a first laser shot applied near to the sperm tail followed by permeabilization with a second laser shot aimed directly at the sperm tail. This sequential approach yielded permanent immobilization at much lower energy values compared with the single shot technique. Following the injection of laser-treated spermatozoa, mouse oocytes underwent normal activation and pronuclear formation. We conclude that a non-contact 1.48 microm diode laser system can be used for immobilization of spermatozoa and for permeabilization of the sperm tail membrane. This laser procedure may offer an alternative to currently used sperm pretreatment prior to intracytoplasmic sperm injection.