We investigate the global principles underlying secretory membrane trafficking, sorting, and compartmentalization within eukaryotic cells. We use live-cell imaging of green fluorescent protein (GFP) fusion proteins in combination with photobleaching and photoactivation techniques to investigate the subcellular localization, mobility, transport routes, and turnover of a variety of proteins with important roles in the organization and regulation of membrane trafficking and compartmentalization. To test mechanistic hypotheses about protein and organelle dynamics, we use quantitative measurements of these protein characteristics in kinetic modeling and simulation experiments. Among the topics under investigation are (i) membrane partitioning and its role in protein sorting and transport in the Golgi apparatus; (ii) biogenesis and dynamics of unconventional organelles; (iii) mitochondrial morphology and its regulation of cell cycle progression; and (iv) cytoskeletal and endomembrane cross-talk in polarized epithelial cells, hematopoietic niche cells, and the developing Drosophila syncytial blastoderm embryo. We also devoted considerable effort to developing new fluorescence microscopy techniques for imaging fluorescently tagged proteins at near-molecular resolution.