The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) regulates cellular motility and the synthesis of organelles and molecules that promote adhesion to a variety of biological and nonbiological surfaces. These properties likely require tight spatial and temporal regulation of c-di-GMP concentration. We have developed genetically encoded fluorescence resonance energy transfer (FRET)-based biosensors to monitor c-di-GMP concentrations within single bacterial cells by microscopy. Fluctuations of c-di-GMP were visualized in diverse Gram-negative bacterial species and observed to be cell cycle dependent. Asymmetrical distribution of c-di-GMP in the progeny correlated with the time of cell division and polarization for Caulobacter crescentus and Pseudomonas aeruginosa. Thus, asymmetrical distribution of c-di-GMP was observed as part of cell division, which may indicate an important regulatory step in extracellular organelle biosynthesis or function.