The orientation of flanking genes may influence the evolution of intergenic regions in which cis-regulatory elements are likely to be located: divergently transcribed genes share their 5' regions, resulting either in smaller "private" spaces or in overlapping regulatory elements. Thus, upstream sequences of divergently transcribed genes (bi-directional upstream regions, or URs) may be more constrained than those of uni-directional gene pairs. We investigated this effect by analyzing nucleotide variation segregating within and between Arabidopsis species. Compared to uni-directional URs, bi-directional URs indeed display lower population mutation rate, as well as more low-frequency polymorphisms. Furthermore, we find that bi-directional regions undergo selection for the maintenance of intergenic distance. Altogether, however, we observe considerable variation in evolutionary rates, with putative signatures of selection on two uni-directional upstream regions.