Genetically altered mice are important research tools for the study of human development and disease. Occasionally, whether or not related to the genetic mutation, mice may become infertile with age and, thus, risk loss of the mutant line. Under conditions in which assisted reproduction techniques (ARTs), such as in vitro fertilization, are unsuccessful, a new strategy, intracytoplasmic sperm injection (ICSI), may be applicable. This technique has been perfected for use in the mouse and is now considered a reliable, effective, and efficient ART. In the study reported here, we "rescued" (i.e., produced offspring, using ICSI from a "last-of-line" mutant male mouse) four lines that otherwise had become infertile and unresponsive to conventional ART's. A total of 26 live pups were produced from eight pregnant recipient foster mothers. Five mutant male mice were derived (one each from three lines, and two from one line), and all survived to adulthood. We found that live born mice could be successfully derived by use of ICSI that subsequently could breed by natural mating to reestablish the mutant line. Because of its effectiveness and reliability under these conditions, ICSI should be considered a powerful addition to the armamentarium of ART's applicable in the genetically-altered mouse, especially when only one male may still be available.