The behavior of the chromophore N-[(1-pyrenylsulfonyl)dipalmitoyl]-L-alpha-phosphatidyleth-anolamine (pyrene-DPPE) embedded in unpolymerized and polymerized air-water monolayers of 10,12-nonacosadiynoic acid (diacetylene 16-8), octadecyl methacrylate (ODMA) and (octadecyldimethylammonio)ethyl methacrylate bromide (ODAEMA), has been investigated by using surface pressure-area isotherm measurements, and steady-state and time-resolved fluorescence spectroscopy. Steady-state fluorescence measurements revealed pyrene-DPPE to be homogeneously distributed in the diacetylene 16-8 monolayer only in the liquid-expanded (LE) phase on an alkaline subphase. The lateral diffusion coefficients determined for the unpolymerized diacetylene 16-8 monolayer from steady-state measurements are in good agreement with literature values. Polymerization of the diacetylene 16-8 monolayer resulted in the formation of a liquid-condensed phase film and aggregation of pyrene-DPPE in the monolayer film, thus preventing a fluorescence quenching study. Pyrene-DPPE was found to be aggregated in both the monomer and polymer monolayers of ODMA. A derivative of ODMA incorporating a quaternary ammonium group (ODAEMA) was synthesized. ODAEMA showed LE isothermal behavior and gave a homogeneous distribution of pyrene-DPPE in the monomer monolayer film. The lateral diffusion coefficients obtained in the unpolymerized ODAEMA monolayer film were found to decrease with increasing surface pressure, reflecting a decrease in monolayer fluidity with compression. In the polymerized film of ODAEMA, nonexponential fluorescence decay behavior of pyrene-DPPE was observed. The fluidity of the polymerized ODAEMA monolayer is estimated to be at least 1 order of magnitude less than that for the corresponding monomer monolayer.