The 5' flanking region of the sea urchin Hemicentrotus pulcherrimus arylsulfatase (Ars) gene was scanned to define cis-regulatory elements required for proper expression congruent to that of the endogenous gene. The region between -100 bp and +38 bp from the transcription start site contains minimum information required for temporal initiation of transcription of the Ars gene. Progressive deletion analysis of Ars-luciferase reporter constructs containing the Ars sequence from -3484 bp to +38 bp suggests the existence of several cis-regulatory elements within this region. Results from luciferase assays of internal deletion mutants show strong enhancer activity detected within the sequence from -194 bp to -144 bp. By gel mobility shift assay, we have identified a nuclear factor that interacts sequence-specifically with this 50 bp region, and appears in a developmental stage-specific manner. Further deletion analysis determined that the enhancer activity lies within a 22 bp sequence between -186 bp and -164 bp.