The transplantation of germ cells into adult recipient gonads is a tool with wide applications in animal breeding and conservation of valuable and/or endangered species; it also provides a means for basic studies involving germ cell (GC) proliferation and differentiation. Here we describe the establishment of a working model for xenogeneic germ cell transplantation (GCT) in sexually competent fish. Spermatogonial cells isolated from juveniles of one species, the pejerrey Odontesthes bonariensis (Atherinopsidae), were surgically transplanted into the gonads of sexually mature Patagonian pejerrey O. hatcheri, which have been partially depleted of endogenous GCs by a combination of Busulfan (40 mg/kg) and high water temperature (25 degrees C) treatments. The observation of the donor cells' behavior showed that transplanted spermatogonial cells were able to recolonize the recipients' gonads and resume spermatogenesis within 6 months from the GCT. The presence of donor-derived gametes was confirmed by PCR in 20% of the surrogate O. hatcheri fathers at 6 months and crosses with O. bonariensis mothers produced hybrids and pure O. bonariensis, with donor-derived germline transmission rates of 1.2-13.3%. These findings indicate that transplantation of spermatogonial cells into sexually competent fish can shorten considerably the production time of donor-derived gametes and offspring and could play a vital role in germline conservation and propagation of valued and/or endangered fish species.