We present the first analysis of human-chimpanzee intron divergence, in which differences in the number of substitutions per intronic site (Ki) can be interpreted as the footprint of different intensities and directions of the pressures of natural selection. Our main findings are as follows: there was a strong positive correlation between intron length and divergence; there was a strong negative correlation between intron length and GC content; and divergence rates vary along introns and depending on their ordinal position within genes (for instance, first introns are more GC rich, longer and more divergent, and divergence is lower at the 3' and 5' ends of all types of introns).We show that the higher divergence of first introns is related to their larger size. Also, the lower divergence of short introns suggests that they may harbor a relatively greater proportion of regulatory elements than long introns. Moreover, our results are consistent with the presence of functionally relevant sequences near the 5' and 3' ends of introns. Finally, our findings suggest that other parts of introns may also be under selective constraints.Introns are neither neutrally evolving sequences nor junk DNA, as they were once considered to be. Increasing amounts of evidence show that they harbor a variety of untranslated RNAs, including microRNAs, small nucleolar RNAs, and guide RNAs for RNA editing [1]. Introns are also important for mRNA processing and transport [2]. Moreover, microarray tiling experiments [3] have shown that a substantial part of the cell's transcriptional activity involves polyadenylated RNA that appears to be derived from intergenic regions, antisense sequences of known transcripts, and introns. Also, recent studies [4,5] show that almost all small nucleolar RNAs and a large proportion of microRNAs in animals are encoded in introns. Finally, novel intronic transcripts are continually being reported (for instance, see the report by Kampa and coworkers [6]), even tho

This research was supported by grants to AN from the Ministerio de Ciencia y Tecnologia (Spain; BOS2003-0870 and BFU2006-15413-C02-01) and the Genome Canada-Genoma España Joint R+D+I Projects in Human Health (JLI/038). OF was supported by a PhD fellowship (SFRH/BD/15856//n2005) from the Fundação para a Ciência e a Tecnologia (Portugal).