The detection of selection, both positive and negative, acting on a DNA sequence or class of nucleotide sites requires comparison with a reference sequence that is unaffected by selection. In Drosophila, recent findings of widespread selective constraint, as well as adaptive evolution, in both coding and noncoding regions highlight the difficulties in choosing such a reference sequence. Here, we investigate the utility of short intron sequences as a reference for the detection of selection. For a set of 119 Drosophila melanogaster genes containing 195 short introns (