Drosophila nuclear introns are commonly assumed to change according to a si
ngle rate of substitution, yet little is known about the evolution of these
non-coding sequences. The hypothesis of a uniform substitution rate for in
trons seems to be at odds with recent findings that the nucleotide composit
ion of introns varies ata scale unknown before, and that their base content
variation is correlated with that of the adjacent exons. However, no direc
t attempt at comparing substitution rates in introns seems to have been add
ressed so far. We have studied the rate of nucleotide substitution over a r
egion of the Xdh gene containing two adjacent short, constitutively spliced
introns, in several species of Drosophila and-related genera. The two intr
ons differ significantly in base composition and substitution rate, with on
e intron evolving at least twice as fast as the other. In addition, the sub
stitution pattern of the introns is positively associated with that of the
surrounding coding regions, evidencing that the molecular evolution of thes
e introns is impacted by the region in which they are embedded. The observe
d differences cannot be attributed to selection acting differently at the l
evel of the secondary structure of the pre-mRNA. Rather, they are better ac
counted for by locally heterogeneous patterns of mutation.