AbstractWe describe a technique for producing germ‐line chimeric rainbow trout, Oncorhynchus mykiss, by microinjection of the isolated blastomeres. FITC‐labeled donor cells and non‐labeled recipient embryos at various developmental stages between the early blastula and early gastrula stages were used for cell transplantation. The chimera formation rate and the degree of donor cell distribution in recipient embryos were evaluated at both the late gastrula stage (5 days post fertilization (dpf)) and the 40‐somite stage (10 dpf). Among the six combinations of developmental stages of donor and recipient embryos, the combination of midblastula (2.5 dpf) donor cells and early blastula (1.5 dpf) recipient embryos gave the highest chimera formation rate and the best distribution pattern of donor cells. Using this combination, chimeric rainbow trout were produced with donor blastomeres from dominant orange‐colored mutant embryos and wild‐type recipient embryos. Of the 238 chimeric embryos produced, 28 (12%) hatched normally and 14 of the 28 fry (50%) had donor‐derived orange body color. To test for germ‐line transmission of donor cells, gametes obtained from the matured chimeras were fertilized with gametes from wild‐type fish. Of the 19 matured chimeras, 6 (32%) yielded donor‐derived orange‐colored progeny, in addition to wild‐type siblings. The contribution rates of donor cells in the germ‐line ranged from 0.3 to 14%. This technique for producing germ‐line chimeras should be a powerful tool for cell‐mediated gene transfer in rainbow trout. Especially, if body color mutants are used for either donor cells or the host embryos, it will be possible to easily concentrate F1 transgenic embryos derived from transplanted donor cells by body color screening. Mol. Reprod. Dev. 59: 380–389, 2001. © 2001 Wiley‐Liss, Inc.