Airway mucus is thought to be required for the clearance of inhaled particles by mucociliary transport, but this view has recently been challenged. To test if mucus is necessary for cilia-driven particle transport, we removed mucus from murine and human ex vivo airway preparations by thorough rinsing with buffer with or without additional dithiothreitol washing. The transport of particles with diameters of 4.5 μm, 200 nm, and 40 nm and of bacteria was analyzed by video microscopy. Complete removal of mucus was verified by wheat germ agglutinin staining and by scanning electron microscopy. In the absence of mucus, we observed efficient transport of particles and bacteria by direct cilia-mediated propulsion or via fluid flow generated by ciliary beating. Virus-sized particles had the tendency to attach to cilia. Because direct contact of particles with ciliated cells occurs in the absence of mucus, we examined if this direct interaction changes epithelial function. Neither bacteria- nor LPS-induced nuclear translocation of NF-κB p65 in ciliated cells occurred, indicating that mere contact between ciliated cells and bacteria during transport does not activate the epithelium. Attachment of virus-sized particles to cilia could induce mucus release and/or increase the ciliary beat frequency. Our results indicate that cilia-driven transport of particles with various sizes is possible in murine and human airways without the presence of mucus. If mucus-free transport fails, the epithelium can react by releasing mucus or increasing the ciliary beat frequency to maintain particle transport.