Based on the authors' previous work an attempt has been made to study water flow in the lipid matrix during pollen hydration. The present study has demonstrated that in the presence of small amounts of water, the type of lipids used defined the time of hydration of pollen in vivo on the stigma and in vitro. Several approaches were used including cryo-scanning electron microscopy, magnetic resonance imaging and Fourier transform infrared microspectroscopic imaging, with the purpose of detecting very small amounts of water. The results show that no water is detectable in the lipid matrix. It was observed and concluded that the water for pollen hydration accumulates as a thin layer at the contact side between pollen and stigma, during the normal process of pollination in plant species with a wet stigma. However, using the same species deprived of the stigma by cell ablation, it was shown that the layer of water observed in wild-type plants is not necessary for pollen hydration