In a recent study, 1373 highly conserved non-coding elements (CNEs) were detected by aligning the human and Takifugu rubripes (Fugu) genomes. The remarkable degree of sequence conservation in CNEs compared with their surroundings suggested comparing the base composition within CNEs with their 5' and 3' flanking regions. The analysis reveals a novel, sharp and distinct signal of nucleotide frequency bias precisely at the border between CNEs and flanking regions.