The Japanese pufferfish (genus Fugu), which possesses a highly compact genome, is becoming a popular model among those interested in sequencing and mapping the genomes of higher vertebrates. Although genomic libraries have been derived and used to study the molecular biology of Fugu, biological material derived from the living organism is difficult to obtain for laboratories distant from the Asian Pacific. We have established cell cultures from two Fugu species: kusafugu, Fugu niphobles, and torafugu, F. rubripes. Cultures derived from F. niphobles fry and F. rubripes eye have been passaged more than 60 times over the course of one year, representing approximately 180 population doublings. Proliferating cultures were also initiated from F. rubripes brain, liver, fin, spleen, kidney, swimbladder, and muscle. Karyotype analyses indicated that F. rubripes eye-derived cells possessed a chromosome number in the diploid range; F. niphobles fry cells were slightly hyperploid. Flow cytometry confirmed that the relative amounts of DNA present in cultured cells from both Fugu species were similar to that measured in blood cells collected from F. rubripes, and approximately one-seventh of that measured in diploid human cells. Telomerase activity was easily detectable in lysates prepared from F. niphobles fry cells and F. rubripes eye cells, consistent with the notion that these cultures are capable of indefinite proliferation.