Fluorescence Resonance Energy Transfer (FRET)- and Fluorescence Lifetime Imaging (FLIM)- based assays have been used to determine the organization of receptor-ligand complexes (polymeric IgA-receptor, pIgA-R; transferrin receptor, TFR; low-density lipoprotein-receptor, LDL-R) in endocytic membranes of MDCK cells. Differently-fluororophore labeled (donor or acceptor) pIgA-R ligands, holo-transferrin (Tfn) and/or LDL were internalized into polarized or non-polarized epithelial MDCK cells, imaged using confocal microscopy and processed for FRET and/or FLIM analysis. A two-parameter confocal FRET assay demonstrates whether receptor-ligand complexes are randomly distributed or clustered. 2-Photon FLIM investigates the effect of the different endocytic environments (e.g. pH, protein composition) on the organization of the receptor-ligand complexes. Our results indicate that receptor clusters show different levels of organization from random to a mixed clustered/random to a clear clustered organization in distinct endocytic compartments. Clusters containing LDL-LDL-R complexes undergo a clustered to random organization which may reflect the release of the LDL from its receptor. Changes in the lifetime distribution of Tfn-TFR complexes localized to different endocytic compartments suggest that these complexes may be exposed to different micro-environments and/or organized in distinct higher-order distributions. Different organizations of receptor-ligand complexes may represent protein sorting and pH-induced conformational changes as they occur during the endosomal trafficking in polarized cells. Analysis of receptor organization in endocytic membranes will provide insights into protein sorting, biogenesis of membrane microdomains and endosomal organization and dynamics.