Using static and dynamic fluorescence we studied the fluorescence properties of a phosphatidylcholine analog of 1,6-diphenyl-1,3,5-hexatriene (DPH-PC) incorporated in lymphocyte plasma membranes with respect to DPH and its cationic derivative 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), in order to study if phospholipid derivatives of DPH may be used to investigate structural and physicochemical properties of specific membrane lipid domains. DPH-PC and TMA-DPH showed similar fluorescence polarization values that were significantly higher with respect to DPH, suggesting a localization of the fluorescent portion of DPH-PC in a more ordered region of the membrane which was probably due to the elecrostatic interactions between phospholipid head-groups. The localization of the fluorescent moiety of DPH-PC near the membrane surface was also supported by the study of the fluorescence decay of the three probes using frequency-domain fluorometry. The main lifetime component of DPH-PC was rather similar to that of TMA-DPH (6.74 versus 6.24, ns) but considerably lower with respect to DPH (10.52 ns), in agreement with data obtained from exponential analysis. In lymphocyte membranes obtained from concanavalin A treated cells, a significant decrease of fluorescence polarization has been shown with DPH and its phosphatidylcholine derivative, but not with TMA-DPH. In liposomes obtained from total lipids extracted from lymphocyte membranes, a decrease of fluorescence polarization has been observed only with DPH. Our results suggest that DPH-PC localizes the fluorescent portion of its molecule in membrane microenvironments of different properties with respect to those probed by DPH and TMA-DPH. The use of DPH-phospholipid derivatives and other DPH-probes may represent an useful tool to study plasma membrane heterogeneity in biological membranes.