Although great progress has been made in identifying microRNAs (miRNAs) and their functions, their essential functional features remain largely unknown. In this study, we systemically investigated the nucleotide and thermodynamic folding distribution characteristics of 3853 miRNAs currently reported for metazoans. We determined that uracil is the dominant nucleotide in both mature and precursor sequences, and that it is particularly enriched at three sites in mature miRNAs: the first, ninth, and the five terminal 3' nucleotides. The location of these enriched uracil nucleotides is particularly interesting because positions one and nine are the edges of the "seed region", which is responsible for targeting mRNAs for gene regulation. The prevalence of U residues at these sites may contribute to the mechanism whereby miRNAs target and bind to their corresponding mRNAs. A comparison of the overall lengths of metazoan pre-miRNAs revealed that they ranged from 53 to 215 nt in length with an average of 88.10+/-14.14 nt, significantly higher than previously reported. Comparisons of miRNA diversity at different taxonomic levels revealed that the 12 features investigated in this study varied significantly among miRNAs represented by different phyla, with particularly high levels of divergence in platyhelminths relative to nematodes, arthropods or vertebrates. By comparison, lower levels of diversity were observed at lower taxonomic levels such that there was a direct relationship between divergence in miRNA features and taxonomic level. We conclude that large-scale genome analysis shows that miRNAs have many more unique features than previously reported. In particular, the distribution of nucleotides suggests an important role for uracil at the boundaries of the 'seed' region and at their termini. These results will facilitate the design of new computational programs for identifying novel miRNAs and investigating the mechanism of miRNA-mediated gene regulation.