The 5' flanking DNA of three related Strongylocentrotus purpuratus genes, Spec1, Spec2a, and Spec2c, were analyzed with respect to structure and cis-regulatory activity. The structural features of DNA sequences upstream of the first intron were highly unusual and implicated certain regions as sites of coordinate control for gene expression. By aligning the genes with a common upstream 600-bp repetitive DNA sequence element, termed RSR, it was shown that a conserved DNA block of approximately 800 bp extended from the 3' end of the first exon to the 5' end of the RSR element. In Spec2a, the conserved sequence block was a continuous stretch of DNA, but in Spec1 and Spec2c, 2.5 to 3 kb of inserted DNA bounded by short direct repeats interrupted the conserved sequence block, thus changing the relative placement of the RSR element and other 5' flanking DNA. Deletion of XhoI fragments containing the 5' half but not the 3' half of the RSR element resulted in a significant decrease in chloroamphenicolacetyl transferase (CAT) activity when Spec-CAT reporter gene fusion plasmids were injected into Lytechinus pictus eggs. These results strongly suggested, but did not prove, that the sequences held in common among the XhoI fragments, that is, the 5' half of the RSR elements, were responsible for the decrease in CAT activity. The Spec2a gene was particularly sensitive to deletions of the XhoI fragment containing the 5' half of the RSR element. The deleted element had several enhancer-like properties when inserted back into various test plasmids: it could be positioned in locations different from the transcriptional start site; in some but not all cases, it could be made to work in the reverse orientation; and it could drive expression of the CAT gene using an SV40 promoter or cryptic promoter elements. These findings suggested that an enhancer-like element important for Spec gene expression was contained within a repetitive DNA sequence. Genomic DNA blots suggested that there are many more of these RSR elements than there are Spec genes.