The sea urchin Strongylocentrotus purpuratus CyIIIb actin gene codes for a cytoskeletal type actin and is activated in the early embryo specifically in the cells destined to become the aboral ectoderm. Deletion constructs of its upstream region fused to the bacterial chloramphenicol acetyltransferase (CAT) gene were expressed in developing embryos following microinjection into eggs. These studies revealed the segments of the upstream region which are necessary for embryonic expression. We mapped the protein:DNA interaction sites in this upstream region by DNase I footprinting. At least five binding sites were identified in the 2173 nucleotide flanking sequence, which seem to include all the necessary elements for quantitative expression. One of these elements (E1) showed different patterns of association in vitro with nuclear proteins isolated from different stages in development. These different interactions are separated when nuclear extracts from ectodermal or endodermal-mesodermal tissues were used. When S. purpuratus eggs were injected with a CAT fusion construct mutated at this site, a decrease in CAT activity was observed only in late stage embryos (plutei), whereas early stage embryos (blastulae) exhibited wild-type CAT activity. These results suggest that the E1 element is involved in the temporal regulation of the CyIIIb gene.