The sperm histone H2B-1 gene of the sea urchin Psammechinus miliaris contains two octamer sequences (ATTTGCAT) and two CCAAT motifs upstream of its TATA box. The CCAAT-binding factors present in nuclear extracts from testis and from blastula and gastrula embryos are indistinguishable by mobility shift and methylation interference analysis. However, there is a testis-specific octamer-binding factor in addition to the ubiquitous form. In DNAase I protection experiments, the CCAAT-binding factor of only the testis extract is able to interact with the sperm H2B promoter. In the two embryonic extracts a novel factor binds with high affinity to sequences overlapping the proximal CCAAT element, thus preventing the DNA interaction of the CCAAT-binding factor in the embryo where the sperm H2B gene is not expressed. This CCAAT displacement protein may therefore act as a repressor of sperm H2B gene transcription.