Previous studies have located some twenty distinct sites within the 2.3 kb 5′ regulatory domain of the sea urchin CyIIIa cytoskeletal actin gene, where there occur in vitro high-specificity interactions with nuclear DNA-binding proteins of the embryo. This gene is activated in late cleavage, exclusively in cells of the aboral ectoderm cell lineages. In this study, we investigate the functional importance in vivo of these sites of DNA-protein interaction. Sea urchin eggs were coinjected with a fusion gene construct in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene is under the control of the entire CyIIIa regulatory domain, together with molar excesses of one of ten nonoverlapping competitor subfragments of this domain, each of which contains one or a few specific site(s) of interaction. The exogenous excess binding sites competitively titrate the available regulatory factors away from the respective sites associated with the CyIIIa.CAT reporter gene. This provides a method for detecting in vivo sites within the regulatory domain that are required for normal levels of expression, without disturbing the structure of the regulatory domain. We thus identify five nonoverlapping regions of the regulatory DNA that apparently function as binding sites for positively acting transcriptional regulatory factors. Competition with a subfragment bearing an octamer site results in embryonic lethality. We find that three other sites display no quantitative competitive interference with CyIIIa.CAT expression, though as shown in the accompanying paper, two of these sites are required for control of spatial expression. We conclude that the complex CyIIIa regulatory domain must assess the state of many distinct and individually necessary interactions in order to properly regulate CyIIIa transcriptional activity in development.