Spfkh1 is a Strongylocentrotus purpuratus transcription factor that contains a winged helix DNA binding domain. Both the gene and overlapping cDNAs encoding this factor have been cloned and completely sequenced. We have mapped the start of transcription by primer extension to a site 600 base pairs 5' to the start of translation. Spfkh1 is transcribed in one open reading frame that contains the DNA binding domain, nuclear localization signal and transactivation domain. The deduced amino acid sequence encodes a 40. 7kDa protein with a pI of 9.96. Alignments of the DNA binding domain with other forkhead domains reveal that this gene falls into Class II of the winged helix transcription factors. We have identified a unique carboxyl-terminal motif of unknown function that is present in all winged helix Class II transcription factors. A phylogenetic analysis of the DNA binding domains shows that, within the Class II, Spfkh1 groups with the deuterostomes as opposed to the protostomes. Analysis of the sequence 5' to the start of translation revealed binding sites for a large number of different transcription factors, many of which are present in multiple copies. The constellation of binding sites in the cis-regulatory region indicates that Spfkh1 is regulated by a complex set of factors, some of which are known to be endoderm specific. Included among these are binding sites for factors downstream of the Wnt/beta-catenin and hedgehog signaling pathways, implicating these pathways in both regulation of Spfkh1 and specification of endoderm.