abstract: Regulation of gene expression is essential for all living cells, and the cell uses a multitude of pathways to ensure proper control of gene expression. One central process in gene expression is mRNA translation; the decoding of mRNA into a protein. A better understanding of translation itself and of translational control requires a method that can visualize translation of single mRNAs in living cells. Here, we report a microscopy-based method that enables visualization of single mRNAs undergoing hundreds of rounds of translation in live cells. This allows us to quantitatively measure initiation and elongation rates of ribosomes on single mRNAs for the first time, revealing a remarkable amount of heterogeneity in their translation dynamics. We further applied this translation imaging method to study gene expression control by small RNAs, which regulate the expression of 1000s of target genes through their association with the effector protein Argonaute (Ago). Agosmall RNA binding to mRNAs results in endonucleolytic cleavage andor translational repression of the target mRNA. Using our imaging method we directly observed the dynamics of target repression by Ago, which revealed that translation of target mRNA potently stimulates target silencing. Taken together, we present here a powerful tool for observing translation of single mRNAs, and for studying translational regulation by small RNAs.