INTRODUCTION:Extracellular vesicles (EVs) represent an important mode of intercellular communication. There is now a growing awareness that predominant EV subtypes; exosomes from endosomal origin, and shed microvesicles from plasma membrane budding, can be further stratified into distinct subtypes, however specific approaches in their isolation and markers that allow them to be discriminated are lacking. Areas covered: Knowledge about these distinct EV subpopulations is important including the regulation of composition, release, targeting/localization, uptake, and function. This review discusses the mechanisms of distinct EV biogenesis and release, defining select EV classes (and subpopulations), which will be crucial for development of EV-based functions and clinical applications. We review the dynamics of cargo sorting leading to the mechanisms of EV heterogeneity, their mechanisms of formation, intracellular trafficking pathways, and provide an uptake about biochemical/functional differences. With advances in purification strategies and proteomic-based quantitation, allows significant benefit in accurately describing differences in EV protein cargo composition and modification. Expert commentary: The advent of quantitative mass spectrometry-based proteomics, in conjunction with advances in molecular cell biology, and EV purification strategies, has contributed significantly to our improved characterization and understanding of the molecular composition and functionality of these distinct EV subpopulations.