To understand how the maternally determined animal-vegetal polarity of the sea urchin embryo is established, we have begun to examine the regulatory apparatus of the gene encoding the Strongylocentrotus purpuratus hatching enzyme (SpHE). Previous studies have shown that the pattern of SpHE mRNA accumulation reflects the animal-vegetal developmental axis in that transcription is strongly upregulated during early cleavage in more animal blastomeres, but not in those around the maternally specified vegetal pole of the 16-cell embryo [Reynolds et al., Development 114, 769-786 (1992)]. Tests of SpHE promoter function in vivo using chloramphenicol acetyltransferase and beta-galactosidase enzymatic reporters define a regulatory region within several hundred nucleotides of the transcription initiation site. This region is sufficient to mediate both strong expression in the early blastula and spatially correct transcription. However, neither this region nor longer upstream sequences are sufficient to reproduce the transcriptional downregulation after very early blastula stage that is observed for endogenous genes. Biochemical assays of protein-DNA interactions within the regulatory region identify at least nine sites binding at least six different factors. These cis elements include Otx (an orthodenticle homologue), CCAAT, ets-related, and three unidentified motifs. Deletions and/or replacements of these cis-elements, alone and in combination, indicate that no single factor is essential for SpHE promoter activity, but instead that various combinations of subsets of these elements are capable of eliciting levels of transcription similar to those of the unaltered regulatory region. This density of regulatory elements is consistent with the intense transcription of endogenous SpHE genes during cleavage.