To evaluate the potential for fertilization by sperm injection into fish eggs, sperm from zebrafish, Danio rerio, were microinjected directly into egg cytoplasm of two different zebrafish lines. To evaluate physiological changes of gametes on the possible performance of intracytoplasmic sperm injection (ICSI), four different combinations of injection conditions were conducted using activated or nonactivated gametes. From a total of 188 zebrafish eggs injected with sperm in all treatments, 31 (16%) developed to blastula, 28 (15%) developed to gastrula, 10 (5%) developed abnormally to larval stages, and another 3 (2%) developed normally and hatched. The highest fertilization rate (blastodisc formation) was achieved by injection of activated spermatozoa into nonactivated eggs (35%). Injections were most effective when performed within the first hour after egg collection. Flow cytometric analysis of the DNA content of the developing ICSI embryos revealed diploidy, and the use of a dominant pigment marker confirmed paternal inheritance. Our study indicates that injection of a single sperm cell into the cytoplasm of zebrafish eggs allows fertilization and subsequent development of normal larvae to hatching and beyond.