Activation of the aboral ectoderm-specific Spec2a gene in blastula-stage sea urchin embryos requires an upstream regulatory region that is part of a repetitive sequence element (RSR) associated with all Spec1/Spec2 genes. Deletions from the 5' end of Spec2a-flanking DNA, monitored for activity using the sea urchin embryo gene-transfer expression system, indicated that this regulatory region has multiple DNA elements and that no positive element lies upstream of the RSR. We mapped the regulatory region using Spec2a fragments containing RSR sequences fused to an SV40 minimal promoter. The region between base pairs -443 and -631, defined as the RSR enhancer, was essential for maximal activity and conferred preferential aboral ectoderm expression to a lacZ reporter gene. Expression was not fully restricted to aboral ectoderm, however, suggesting that negative spatial elements are also associated with the proper activation of Spec2a. DNaseI footprinting and band-shift analysis of the RSR enhancer identified an A/T-rich DNA element, the A/T palindrome. This element binds a single 45-kDa nuclear protein, the A/T palindrome binding protein (A/TBP), whose specificity suggests a possible relationship with the bicoid-class homeodomain proteins. Mutations of the A/T palindrome are incapable of binding the 45-kDa protein and lower promoter activity by eight-fold. DNA-binding activity for A/TBP is low in unfertilized eggs, increases by the 16-cell stage and continues rising in blastulae. These data suggest that A/TBP plays a major role in the activation of the Spec2a gene in aboral ectoderm cells.