Pyrene and pyrene-labeled phosphatidylcholine (PyC(10)PPC) were used as fluorescent probes embedded in behenic acid/behenate and in dimyristoyl-L-alpha-phosphatidic acid/dimyristoyl-L-alpha-phosphatidylcholine (DMPA/DMPC) monolayers and related Langmuir-Blodgett films (LB films). The influence of the probe on the integrity of the lipid monolayer was first controlled through the characteristics of surface pressure-molecular area isotherms. The characterization of LB films was based on the transfer ratio values and on the type of multilayers. Steady-state fluorescence studies performed on the corresponding multilayers enabled the advantages and drawbacks of each probe to be determined. Pyrene does not alter the molecular packing of the phospholipidic matrix but is not transferred in a sufficient amount to give a detectable signal. On the contrary, excellent results were obtained with PyC(10)PPC, which can be used at a molar fraction lower than 4.2%, avoiding any disturbance both in the integrity of the monolayer and in the stacking of the transferred layers in LB films.