ABSTRACT: The zebrafish has become an important model for cancer research. Several cancer models have been established by transgenic expression of human or mouse oncogenes in zebrafish. Since it is amenable to efficient transgenesis, zebrafish has immense potential to be used for studying interaction of oncogenes and pathways at the organismal level. Using the Gal4VP16-UAS binary transgenic expression approach, we established stable transgenic lines expressing an EGFP tagged activated zebrafish smoothened (smoa1). Expression of the zebrafish smoa1 itself did not lead to tumor formation either in founder fish or subsequent generations. Neither did expression of a constitutively active form of human AKT1. However, co-expression of the two oncogenes resulted in several tumor types, including spindle cell sarcoma, rhabdomyoma, ocular melanoma, astrocytoma and so on. All tumor types showed GFP expression, suggesting involvement of zebrafish smoa1 in tumorigenesis. Immunofluorescence studies showed that tumors also expressed elevated levels of phosphorylated AKT, indicating activation of the PI3K-AKT pathway. These results suggest that co-activation of the hedgehog and AKT pathways promote tumorigenesis, and the binary transgenic approach may become an invaluable tool for studying interaction of oncogenes and oncogenic pathways in zebrafish.