MicroRNAs (miRNAs) are small endogenous RNAs that pair to sites in mRNAs to direct post-transcriptional repression. Many sites that match the miRNA seed (nucleotides 2–7), particularly those in 3' untranslated regions (3'UTRs), are preferentially conserved. The authors developed a new tool to detect preferentially conserved miRNA target sites, which increased the number of detected sites by nearly threefold. The new tool more efficiently incorporates new genomes and better controls for background conservation by accounting for mutational biases, dinucleotide conservation rates, and the conservation rates of individual UTRs. It detected a new site type, the "offset 6mer," and found that over 45,000 miRNA target sites in human 3'UTRs are conserved above background levels, with >60% of human protein-coding genes under selective pressure to maintain pairing to miRNAs. Mammalian-specific miRNAs have far fewer conserved targets than broadly conserved miRNAs. Although pairing to the 3' end of miRNAs can compensate for seed mismatches, this class of sites constitutes less than 2% of all preferentially conserved sites. The new tool enables statistically powerful analysis of individual miRNA target sites, with the probability of preferentially conserved targeting (P_CT) correlating with experimental measurements of repression. The authors' expanded set of target predictions, including conserved 3'-compensatory sites, are available at the TargetScan website, which displays the P_CT for each site and each predicted target. The study shows that miRNAs are ~22-nucleotide endogenous RNAs that derive from distinctive hairpin precursors. After incorporation into a silencing complex, miRNAs can pair to mRNAs and specify post-transcriptional repression. The search for biological targets of metazoan miRNAs has benefited from comparative analysis of orthologous mRNAs. Targets can be predicted by requiring conserved Watson–Crick pairing to the 5' region of the miRNA, known as the miRNA seed. The four types of seed-matched sites are the 6mer, 7mer-m8, 7mer-A1, and 8mer. The 8mer is the most effective, followed by 7mer-m8, 7mer-A1, and 6mer. Comparative analysis revealed that many genes of mammals, flies, and worms are miRNA targets. Over one third of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs. The selective depletion of seed-matching sites in messages highly expressed in the same tissues as the miRNAs implies frequent nonconserved targeting. The study used a branch-length metric to evaluate motif conservation, building a phylogenetic tree based on genomic regions under investigation. The method controlledMicroRNAs (miRNAs) are small endogenous RNAs that pair to sites in mRNAs to direct post-transcriptional repression. Many sites that match the miRNA seed (nucleotides 2–7), particularly those in 3' untranslated regions (3'UTRs), are preferentially conserved. The authors developed a new tool to detect preferentially conserved miRNA target sites, which increased the number of detected sites by nearly threefold. The new tool more efficiently incorporates new genomes and better controls for background conservation by accounting for mutational biases, dinucleotide conservation rates, and the conservation rates of individual UTRs. It detected a new site type, the "offset 6mer," and found that over 45,000 miRNA target sites in human 3'UTRs are conserved above background levels, with >60% of human protein-coding genes under selective pressure to maintain pairing to miRNAs. Mammalian-specific miRNAs have far fewer conserved targets than broadly conserved miRNAs. Although pairing to the 3' end of miRNAs can compensate for seed mismatches, this class of sites constitutes less than 2% of all preferentially conserved sites. The new tool enables statistically powerful analysis of individual miRNA target sites, with the probability of preferentially conserved targeting (P_CT) correlating with experimental measurements of repression. The authors' expanded set of target predictions, including conserved 3'-compensatory sites, are available at the TargetScan website, which displays the P_CT for each site and each predicted target. The study shows that miRNAs are ~22-nucleotide endogenous RNAs that derive from distinctive hairpin precursors. After incorporation into a silencing complex, miRNAs can pair to mRNAs and specify post-transcriptional repression. The search for biological targets of metazoan miRNAs has benefited from comparative analysis of orthologous mRNAs. Targets can be predicted by requiring conserved Watson–Crick pairing to the 5' region of the miRNA, known as the miRNA seed. The four types of seed-matched sites are the 6mer, 7mer-m8, 7mer-A1, and 8mer. The 8mer is the most effective, followed by 7mer-m8, 7mer-A1, and 6mer. Comparative analysis revealed that many genes of mammals, flies, and worms are miRNA targets. Over one third of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs. The selective depletion of seed-matching sites in messages highly expressed in the same tissues as the miRNAs implies frequent nonconserved targeting. The study used a branch-length metric to evaluate motif conservation, building a phylogenetic tree based on genomic regions under investigation. The method controlled