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Publisher Summary This chapter discusses the use of temperature experiments in studies of protein transport in the exocytic pathway with emphasis on the description of immunocytochemical techniques used in protein detection in light and electron microscropy. The availability of well-characterized soluble and membrane-bound markers made it possible to discover thermosensitive steps in the endocytic pathway. Studies of virus glycoprotein transport have initiated the analysis of the function of distinct intracellular sites in the biosynthetic pathway at which protein movement is arrested at reduced temperatures. Temperature experiments with cultured cells grown on plastic or on glass coverslips require simple equipment. Incubation is most conveniently carried out in water baths adjusted to the appropriate temperature. Bath temperature can be easily monitored with a thermometer punched through the Styrofoam cover, and adjusted, if necessary, by adding a few chucks of ice. At 15°C proteins accumulate in a compartment between the endoplasmic reticulum (ER) and the Golgi complex, whereas at 20°C accumulation occurs at the level of a late-Golgi compartment. The reversible temperature-induced transport blocks provide valuable tools for the morphological and biochemical analysis of membrane traffic. Distinct threshold temperatures exist for different steps in transport pathways, but temperature reduction can also be used to slow down events among different sites and to increase resolution.