The HNF-3/HFH-4/Foxj1, a transcription factor, has been reported to be involved in systemic autoimmunity and cilia genesis in vertebrates. The zebrafish genome expressed two paralogous foxj1 genes, foxj1a and foxj1b. In this study, we demonstrate that down-regulation of either foxj1a or foxj1b by injecting antisense morpholino at the one-cell stage results in randomized expression of the early left-right (LR) asymmetric markers lefty2, southpaw, pitx2c and the later internal organ markers tpm4-tv1, cmlc2, cp in zebrafish embryos. Overexpression of foxj1a and foxj1b by injecting synthetic mRNAs also disrupts normal LR asymmetries. These data indicate that the two foxj1 genes are required for normal laterality development in zebrafish embryos. In contrast to foxj1b knockdown exclusively in dorsal forerunner cells (DFCs) that has little effect on laterality, foxj1a knockdown in DFCs randomizes the LR patterns of the markers. Thus, foxj1a regulates asymmetric development through DFCs in a cell-autonomous fashion but foxj1b functions indirectly.