Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. In this report, AECC member Singer and colleagues describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. He generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3' untranslated region of the essential betabeta-actin gene. As beta-actin-MBS was ubiquitously expressed, he could uniquely address endogenous mRNA regulation in any tissue or cell type. He simultaneously followed transcription from the beta-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. He tracked single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ hybridization (FISH), allowing detection and localization of single beta-actin mRNA molecules in various mouse tissues.