Time-resolved fluorescence measurements on liposomes prepared with 1 mol% pyrene-labeled lipids (PLLs) with a headgroup bearing either an alcohol (PSOH) or an imido diacetic acid (PSIDA) and 99 mol% 1-palmitoyl-2-oleyl-3-sn-phosphatidylcholines (POPC) or 99 mol% distearylphosphatidylcholine (DSPC) were performed to investigate how lipids phase separate within the membrane bilayer. Global analysis of the fluorescence decays with the fluorescence blob model (FBM) led to the conclusion that the PLLs were homogeneously distributed on the surface of POPC vesicles while the PLLs phase-separated in the DSPC vesicles. The analysis yielded the fraction of aggregated pyrenes, f(agg). The large f(agg) values found for PSIDA suggest that the imido diacetic acid headgroup of PSIDA induces self-aggregation and phase separation in both membranes. The addition of external cations such as Cu2+ and La3+ was shown to hinder diffusional encounters between PSIDAs. The cations seem to target preferentially unassociated PSIDAs rather than aggregated PSIDA clusters. Accounting for the quenching of pyretic. by Cu2+ enables one to use PSIDA to probe the microviscosity of the lipid membrane. Using this effect, the environment of PSIDA in the DSPC membrane was found to be about 6 times more viscous than that in the POPC membrane. This difference is at to the difference in viscosity of the fluid POPC membrane and the gel-like DSPC membranes.