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The cytoplasmic membrane compartmentalises the bacterial cell into cytoplasm and periplasm. Proteins located in this membrane have a defined topology that is established during their biogenesis. However, the accuracy of this fundamental biosynthetic process is unknown. We developed compartment-specific fluorescence labelling methods with up to single-molecule sensitivity. Application of these methods to the single and multi-spanning membrane proteins of the Tat protein transport system revealed rare topogenesis errors. This methodology also detected low level soluble protein mislocalization from the cytoplasm to the periplasm. This study shows that it is possible to uncover rare errors in protein localization by leveraging the high sensitivity of fluorescence methods., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ‘Author Achillefs Kapanidis has a financial interest in Oxford NanoImager which manufactures the fluorescence microscope used in this work. Author Achillefs Kapanidis is a member of the Editorial Board of the Journal of Molecular Biology.’, (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)