The TGFbetas, a family of secreted polypeptide growth factors, are critical regulators of mammalian orofacial development. The importance of the TGFbetas in development of the orofacial region in mice is underscored by the resulting orofacial clefts in mice with targeted deletion of either TGFbeta2 or TGFbeta3 and most recently, a conditional knockout of the type II TGFbeta receptor (TbetaRII) gene. The TGFbetas signal via binding to specific cell surface receptors which, in turn, activates translocation of the nucleocytoplasmic Smad transcriptional regulators. Smads 2 and 3 are TGFbeta-specific transcriptional regulators that bind DNA through their conserved MH1 domains and activate or inhibit transcription of TGFbeta-responsive genes through their MH2 domains. To search for novel Smad binding proteins expressed in developing murine orofacial tissue, a yeast two-hybrid assay was utilized to screen a cDNA expression library constructed from fetal murine orofacial tissue. Several novel Smad binding proteins were identified. These include a putative zinc finger protein (ZNF198), peroxisomal biogenesis factor 6 (Pex6), eucaryotic translation initiation factor 4E nuclear import factor 1 (4-ET), and splicing factor 3b subunit 2 (SF3b2). Results of the yeast two-hybrid screen were verified by GST pull-down assays which confirmed the interaction of these proteins with the MH2 domain of Smad 3, and also indicated interaction of these proteins with additional Smad family members. The identification of these proteins as Smad binding partners allows exploration of new mechanisms whereby TGFbeta signaling may be regulated, and reveals additional potential interactions with other signaling pathways.