Nitrous oxide gas (N(2)O) can be used to produce polyploid plants, but the mechanism of action is unknown. The actin and microtubule cytoskeleton was observed in N(2)O-treated microsporocytes of Lilium spp 'Asiatic hybrid lilies' using fluorescence microscopy after staining with DAPI, FITC-conjugated tubulin antibody, and phalloidin-conjugated Alexa Fluor 546. Additionally, microsporocytes of L. longiflorum were observed with acetocarmine staining following N(2)O treatment. A typical metaphase I microtubule distribution was observed in control microsporocytes. After treatment with N(2)O for 24 h, microtubules were effectively depolymerized; this prevented chromosomes from moving to the poles, resulting in chromosome retention in the center of N(2)O-treated cells. Cell plate formation took place without delay, however, yielding one daughter cell with a diploid genome and another daughter without chromosomes. In addition, N(2)O treatment often induced micronuclei due to aberrant chromosome separation during cytokinesis. Actin filaments in microsporocytes are insensitive to N(2)O. These findings indicate that N(2)O mediates polyploidization by inhibiting microtubule polymerization, but not actin filament formation, during microsporocyte meiosis.