Small noncoding RNAs as well as folded RNA structures in genic regions are crucial for many cellular processes. They are involved in posttranscriptional gene regulation (microRNAs), RNA modification (small nucleolar RNAs), regulation of splicing, correct localization of proteins, and many other processes. In most cases, a distinct secondary structure of the molecule is necessary for its correct function. Hence, selection should act to retain the structure of the molecule, although the underlying sequence is allowed to vary. Here, we present the first genome-wide estimates of selective constraints in folded RNA molecules in the nuclear genomes of drosophilids and hominids. In comparison to putatively neutrally evolving sites, we observe substantially reduced rates of substitutions at paired and unpaired sites of folded molecules. We estimated evolutionary constraints to be in the ranges of (0.974,0.991) and (0.895,1.000) for paired nucleotides in drosophilids and hominids, respectively. These values are significantly higher than for constraints at nonsynonymous sites of protein-coding genes in both genera. Nonetheless, valleys of only moderately reduced fitness (s ≈ 10(-4)) are sufficient to generate the observed fraction of nucleotide changes that are removed by purifying selection. In addition, a comparison of selective coefficients between drosophilids and hominids revealed significantly higher constraints in drosophilids, which can be attributed to the difference in long-term effective population size between these two groups of species. This difference is particularly apparent at the independently evolving (unpaired) sites.