Transforming growth factor-beta (TGF-beta3) gene disruption causes cleft secondary palate. Pax9 and Sonic hedgehog (Shh) genes are involved in the patterning of vertebrate embryonic tissues, including the facial skeleton. We investigated the expression of Pax9 and Shh genes during normal mouse palate development and in the developing cleft palates of TGF-beta3 null embryos. Whole mount in situ hybridization was conducted with use of Pax9 and Shh riboprobes for TGF-beta3 null, heterozygous and wild type mice at E12.5-E16.5. Histological analysis was processed by section in situ hybridization. In the wild type, Pax9 and Shh were expressed in the palate between E12.5-E15.5. Shh expression in the secondary palate was restricted to the rugae and the soft palate. Pax9 expression was predominantly in the palatal medial edge between E14.5 and E15.5. These patterns suggest that Shh and Pax9 may have different functions during palate development. In TGF-beta3 null mice, both genes expression patterns in the palate were different to those in wild type mice. In TGF-beta3 null mice, Pax9 expression was much reduced in the palatal medial edge at the critical time of palatal fusion (E14.5-E15.5). Shh expression in the palates of TGF-beta3 null mice was reduced throughout E12.5-E15.5, whilst Shh expression in heterozygous did not appear down regulated compared with the wild type. These results indicate that Pax9 and Shh expression are altered when the TGF-beta3 gene is deleted and suggest that Pax9 and Shh may be involved in the TGF-beta3 regulation of normal palatal fusion.