The class of small RNAs known as microRNAs (miRNAs) has a demonstrated role in the negative regulation of gene expression in both plants and animals. These small molecules have been shown to play a critical role in a wide range of developmental and physiological pathways. Although hundreds of different miRNAs have now been identified using cloning and computational approaches, characterization of their targets and biological roles has been more limited. New sequencing technologies promise to accelerate the sequencing of small RNAs and additional genetic and genomic strategies are being applied to assess their regulatory function on RNA targets. These technologies will enable the identification of large numbers of small RNAs from diverse species, and comparative genomics approaches based on these data are likely to identify additional miRNAs. Combined with bioinformatics and experimental approaches to separate miRNAs from short-interfering RNAs (siRNAs), the pace of miRNA discovery is likely to accelerate, leading to an improved understanding of miRNA function and biological significance.