The SpFoxB gene is transiently expressed first in the mesoderm, then in the endoderm and oral ectoderm during sea urchin gastrulation. Perturbations of a number of proteins involved in endomesoderm specification have been shown to alter the mRNA levels of SpFoxB, but the cis-regulatory elements required for expression of SpFoxB have not been examined. In order to investigate this, we have screened the SpFoxB gene for sequences that can drive its expression. Both positive and negative cis-regulatory elements were found to be present. An enhancer was found that contains four GATA sites and four YY1 sites clustered within 210 base pairs (bp), as well as three lef/tcf binding sites. Electrophoretic mobility shifts indicate that the lef/tcf sites bind a complex of proteins that include beta-catenin in early cleavage, but not during subsequent stages of development. The GATA and YY1 sites bind nuclear proteins prior to SpFoxB transcription, and this binding diminishes coincident with cessation of transcription. Deletion of the GATA/YY1 sites causes a significant decrease in transcription. The DNA binding site of the SpFoxB protein has been determined, and Fox binding sites are found within the 5' UTR of SpFoxB.