PTEN is a tumor suppressor gene associated with multiple tumor types. PTEN function is essential for early embryonic development and is involved in the regulation of cell size, number, and survival. By dephosphorylating PIP(3), PTEN normally acts to inhibit the PI3-Kinase/AKT pathway. Here we have identified two zebrafish orthologs, ptena and ptenb, of the single mammalian PTEN gene and analyzed the role of these genes in zebrafish development. Ptena transcripts were expressed throughout the embryo at early somitogenesis. By 24 hpf, expression was predominant in the central nervous system, axial vasculature, retina, branchial arches, ear, lateral line primordium, and pectoral fin bud. Ptenb was also ubiquitously expressed early in somitogenesis, but transcripts became more restricted to the somites and central nervous system as development progressed. By 48 hpf, ptena and ptenb were expressed predominantly in the central nervous system, branchial arches, pectoral fins, and eye. Antisense morpholinos were used to knock down translation of ptena and ptenb mRNA in zebrafish embryos. Knockdown of either pten gene caused increased levels of phosphorylated Akt in morphant embryos, indicating that Ptena and Ptenb each possess PIP(3) lipid phosphatase activity. Ptena morphants had irregularities in notochord shape (73%), vasculogenesis (83%), head shape (72%), and inner ear development (59%). The most noticeable defects in ptenb morphants were upward hooked tails (73%), domed heads (83%), and reduced yolk extensions (90%). These results indicate that ptena and ptenb encode functional enzymes and that each pten gene plays a distinct role during zebrafish embryogenesis.