Bafilomycin A₁ is a specific inhibitor of the vacuolar type H+-ATPase (V-ATPase) in cells, and inhibits the acidification of organelles containing this enzyme, such as lysosomes and endosomes. Recently, while editing and reviewing chapters on autophagy for Methods in Enzymology, we noticed repeated references to the effect of bafilomycin A₁ in blocking the fusion of autophagosomes with lysosomes. Of course we have seen this in various research papers as well, but reading this routinely in chapters written by various people over a short period of time really caused this to stand out. Every one of these chapters referred to the paper by Yamamoto et al. In that paper, treatment with 100 nM bafilomycin A₁ for 1 h blocks the fusion of autophagosomes with lysosomes in the rat hepatoma H-4-II-E cell line, based on data from electron microscopy. However, data from one of our labs noted an apparently different result in a relatively recent manuscript. Therefore, we decided to look into this more carefully.