Summary: SOX3 is one of the earliest neural markers in vertebrates and is thought to play a role in specifying neuronal fate. To investigate the regulation of Sox3 expression we identified cis-regulatory regions in the Sox3 promoter that direct tissue-specific heterologous marker gene expression in transgenic mice. Our results show that an 8.3 kb fragment, comprising 3 kb upstream and 3 kb downstream of the Sox3 transcriptional unit, is sufficient in a lacZ reporter construct to reproduce most aspects of Sox3 expression during CNS development from headfold to midgestation stages. The apparently uniform expression of Sox3 in the neural tube depends, however, on the combined action of distinct regulatory modules within this 8.3 kb region. Each of these gives expression in a subdomain of the complete expression pattern. These are restricted along both the rostral-caudal and dorso-ventral axes and can be quite specific, one element giving expression largely confined to V2 interneuron precursors. We also find that at least some of the regulatory sequences are able to drive expression of the transgene in the CNS Xenopus laevis embryos in a manner that reflects the endogenous Sox3 expression pattern. These results imply that the underlying mechanism regulating early CNS patterning is conserved, despite several substantial differences in neurogenesis between mammals and amphibians. genesis 36:12–24, 2003. © 2003 Wiley-Liss, Inc.