The presence and localization of actin and myosin have been examined in pollen tubes of Lilium longiflorum and Nicotiana alata. Immunoblot analysis of pollen tube extracts with antibodies to actin, myosins IA and IB, myosin II, and myosin V reveals the presence of these contractile proteins. Immunofluorescence microscopy using various methods to preserve the pollen tubes; chemical fixation, rapid freeze fixation and freeze substitution (RF-FS) followed by rehydration or by embeddment in a methacrylate mixture, was performed to optimize preservation. Immunocytochemistry reaffirmed that actin is localized longitudinally in the active streaming lanes and near the cortical surface of the pollen tube. Myosin I was localized to the plasma membrane, larger organelles, the surface of the generative cell and the vegetative nucleus, whereas, myosin V was found in the vegetative cytoplasm in a punctate fashion representing smaller organelles. Myosin II subfragment 1 and light meromyosin were localized in a punctate fashion on the larger organelles throughout the vegetative cytoplasm. In addition, isolated generative cells and vegetative nuclei labeled only with the myosin I antibody. Competition studies indicated the specificity of the heterologous antibodies utilized in this study suggesting the presence of three classes of myosins in pollen. These results lead to the following hypothesis: Myosin I may move the generative cell and vegetative nucleus unidirectionally through the pollen tube to the tip, while myosin V moves the smaller organelles and myosins I and II move the larger organelles (bidirectionally) that are involved in growth.