In this paper, we report the monolayer behavior of pure (5-n-hexadecanoylamino)fluorescein (HDFL) at the air-water interface. The spectroscopic characteristics of pure HDFL films at the air-water interface and the mixed Langmuir-Blodgett (LB) films of HDFL and palmitic acid (PA) deposited on quartz substrates have been studied and compared with the spectroscopic characteristics of HDFL in solution. The absorption and emission spectra of HDFL at the air-water interface were similar to those in polar, protic solvents but completely different in aprotic, apolar solvents. Detailed spectroscopic studies indicate that in protic solvents the fluorescein moieties exist as cations and zwitterions while in nonpolar environments the fluorescein chromophore exists as an inner lactone that is nonfluorescent. Spectroscopic studies of the mixed films of HDFL and PA transferred on quartz substrates provide evidence of aggregation and formation of at least two different aggregated species, one of them being fluorescent while the other is not. The large differences in the spectral characteristics between the monolayers at the air-water interface and the transferred layers indicate the existence of HDFL moieties in different molecular configurations that are microenvironment dependent. Surface pressure dependent steady-state in, situ fluorescence studies confirm that fluorescence quenching of the dye with increasing surface pressure occurs as a result of efficient energy transfer from the fluorescent monomeric species to the aggregates that are likely nonfluorescent and decay therefrom by nonradiative processes. This study establishes that fluoresceinated dyes may be utilized as efficient molecular probes for ascertaining polarity in supramolecular assemblies.