In this paper we present a structural and functional characterization of a new sea urchin embryo transcription factor, SpRunt-1. This factor was isolated by means of its specific interaction with a cis-regulatory target site of the CyIIIa gene. Here we show that this target site, the P7I site, is required for normal embryonic activation of CyIIIa x CAT reporter gene constructs. An oligonucleotide affinity column bearing the P7I target site purifies a 21-kDa polypeptide from blastula-stage nuclear extracts, and the amino acid sequence obtained from this polypeptide was used to generate a nucleic acid probe with which the corresponding cDNA was cloned. The cDNA encodes an approximately 60-kDa protein, SpRunt-1, which includes a "runt domain" that is closely homologous to those of Drosophila and mammalian runt domain transcription factors. RNA and genomic blots show that SpRunt-1 is represented by a single embryonic transcript, encoded by one of possibly two runt-domain-containing genes. By RNA probe protection we found that transcripts of SpRunt-1 increase in concentration dramatically after the blastula stage of development, suggesting that the up-regulation of CyIIIa that occurs after blastula stage is a function of zygotically transcribed SpRunt-1. These results are discussed with reference to known features of the runt domain family of transcription factors.