The interaction of β-cyclodextrin (β-CD) with different lipids has been studied, using Langmuir monolayers kept at constant surface pressure or constant spreading surface. Results show that β-CD, injected beneath the monolayer, is able to desorb unsaturated palmitoyloleoylphosphatidylcholine (POPC) and sphingomyelin (SM) under specific experimental conditions. In this last case, SM monolayers, labeled with the fluorescent NBD-PC probe, were also observed by fluorescence microscopy, before and after β-CD injection. Images show that SM monolayers are more homogeneous after β-CD injection, because of the lipid desorption. At last, it seems that lipid desorption occurs only in a restricted surface pressure range, depending on the lipid.