Early development of the embryo is directed by maternal gene products and characterised by limited zygotic gene activity, cell division synchrony and no cell motility in several vertebrates including fish and frogs. At the midblastula transition (MBT), zygotic transcription is grossly activated, cells become motile and cell divisions become asynchronous. The aim of this study was to identify genes whose expression is up-regulated at the MBT in zebrafish. Suppression subtractive hybridisation (SSH) was employed to isolate 48 unique cDNAs, 28 of which show significant similarity to known genes and 20 represent novel cDNAs. Twenty one of these genes, with potential roles in transcriptional regulation, cell cycle control, and embryonic patterning showed increased expression at the MBT. Our results demonstrate the value of SSH as a tool to clone novel, zygotic, developmentally regulated genes that may be important in the progression of the MBT and embryonic patterning.