Expression of the CyIIIa cytoskeletal actin gene is a marker of differential gene activation in the aboral ectoderm of the early sea urchin embryo. Gene transfer experiments have defined a 2,300 nucleotide cis-regulatory domain required for the correct spatial and temporal control of this gene. This domain includes at least 20 sites at which relatively stable DNA--protein complexes form in vitro on reaction with embryo nuclear extracts. We report the nucleotide sequence of the whole regulatory domain and map the sites at which high-specificity DNA--protein interactions occur. These were located initially by gel shift assays carried out on progressive restriction digests of given subfragments of the large regulatory domain and were located more exactly by oligonucleotide gel shift competitions. Eight of the sites of specific interaction are unique within the CyIIIa regulatory domain, and the remainder consist of five different sites that occur more than once. We observe some well known sequences also found in regulatory regions of other genes, e.g., "CCAAT" and "octamer" elements. The various sites have been classified regarding putative biological function in other work, and the present studies permit an assessment of the number and complexity of interactions constituting each functional class and of the relative locations of sites of each class.