We herein report a reexamination of Langmuir and Langmuir-Schaefer films of (5-octadecanoylamino)-fluorescein (ODFL) prepared from three different spreading solvents: ethanol (EtOH), CHCl3/EtOH (9:1, v/v), and CHCl3/trifluoroacetic acid (9:1, v/v). It was found that the limiting molecular area of the ODFL monolayer is strongly spreading solvent-dependent, ranging from 15 to 64 Angstrom (2)molecule(-1). These results strongly disgree with reported results from Tsukanova et al. (Langmuir 2000, 16, 1167) of the same molecule by using ethanol as spreading solvent. Our study also shows that this fluorescein amphiphile significantly aggregates at the air-water interface. The UV-vis spectra of the monolayer are redshifted about 20 nm compared to that in the solution. Fluorescence quenching was observed from both a pure ODFL monolayer and a monolayer of ODFL mixed with stearic acid. However, fluorescence emission spectra were readily observed from mixed monolayers of ODFL with three synthetic peptide lipid molecules. This study shows that one should be extremely cautious when mixing fluorescence lipids as probes for Langmuir and Langmuir-Blodgett film investigations.