MicroRNAs (miRNAs) are small noncoding RNAs that may target more than one-third of human genes, yet the mechanisms used by miRNAs to repress translation of target mRNAs are obscure. Using a recently described cell-free assay of miRNA function, we observe that miRNA-targeted mRNAs are enriched for 40S but not 60S ribosome components. Additionally, toeprinting analysis of miRNA-targeted mRNAs demonstrates that approximately 18 nt 3' relative to the initiating AUG are protected, consistent with 40S ribosome subunits positioned at the AUG codon. Our results suggest that miRNAs repress translation initiation by preventing 60S subunit joining to miRNA-targeted mRNAs.