In human cancer, PTEN (Phosphatase and TENsin homolog on chromosome 10, also referred to as MMAC1 and TEP1) is a frequently mutated tumor suppressor gene. We have used the zebrafish as a model to investigate the role of Pten in embryonic development and tumorigenesis. The zebrafish genome encodes two pten genes, ptena and ptenb. Here, we report that both Pten gene products from zebrafish are functional. Target-selected inactivation of ptena and ptenb revealed that Ptena and Ptenb have redundant functions in embryonic development, in that ptena-/- and ptenb-/- mutants did not show embryonic phenotypes. Homozygous single mutants survived as adults and they were viable and fertile. Double homozygous ptena-/-ptenb-/- mutants died at 5 days post fertilization with pleiotropic defects. These defects were rescued by treatment with the phosphatidylinositol-3-kinase inhibitor, LY294002. Double homozygous embryos showed enhanced cellular proliferation. In addition, cell survival was dramatically enhanced in embryos that lack functional Pten upon gamma-irradiation. Surprisingly, adult ptenb-/- zebrafish developed ocular tumors later in life, despite the expression of ptena in adult eyes. We conclude that whereas Ptena and Ptenb have redundant functions in embryonic development, they apparently do not have completely overlapping functions later in life. These pten mutant zebrafish represent a unique model to screen for genetic and/or chemical suppressors of Pten loss-of-function.