The organization of the normal airway mucus system differs in small experimental animals from that in humans and large mammals. To address normal murine airway mucociliary clearance, Alcian blue-stained mucus transport was measured ex vivo on tracheal tissues of naïve C57BL/6, Muc5b–/–, Muc5ac–/–, and EGFP-tagged Muc5b reporter mice. Close to the larynx with a few submucosal glands, the mucus appeared as thick bundles. More distally in the trachea and in large bronchi, Alcian blue-stained mucus was organized in cloud-like formations based on the Muc5b mucin. On tilted tissue, the mucus clouds moved upward toward the larynx with an average velocity of 12 μm/s compared with 20 μm/s for beads not associated with clouds. In Muc5ac–/– mice, Muc5b formed mucus strands attached to the tissue surface, while in Muc5b–/– mice, Muc5ac had a more variable appearance. The normal mouse lung mucus thus appears as discontinuous clouds, clearly different from the stagnant mucus layer in diseased lungs. [ABSTRACT FROM AUTHOR]