In situ hybridization methods have proven to be useful for studying mRNAs within intact embryos. However, analysing multiple mRNAs and achieving quantitative signals while overcoming background fluorescence has posed significant challenges. The recent development of multiplexed quantitative signal amplification using hybridization chain reaction (HCR) has overcome some of these obstacles. Now, Niles Pierce and colleagues report a novel ‘third-generation’ in situ HCR approach in which background signals are significantly supressed. Their new method uses pairs of split-initiator probes to detect target mRNAs: unlike standard in situ HCR probes, which can bind non-specifically within the specimen and trigger HCR to generate amplified background, split-initiator probes each carry half of the HCR initiator sequence and thus only trigger HCR when they both bind specifically to their target. This automatic background suppression property is maintained during signal amplification as HCR hairpins are kinetically trapped, ensuring that any that bind non-specifically are incapable of triggering spurious chain reactions. The authors show that in situ HCR v3.0 in whole-mount chicken embryos dramatically increases signal-to-background levels but also removes the need for probe set optimization. Finally, they show that this method allows mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount embryos and via high-throughput flow cytometry, and mRNA absolute quantitation via single-molecule imaging in thick autofluorescent samples. Overall, this new approach promises to be a valuable tool for the study of gene expression.