SNARE proteins control the membrane fusion events of membrane trafficking pathways. Work in epithelial cells has shown that polarized trafficking to the apical and basolateral plasma membrane domains requires different sets of SNAREs, suggesting a mechanism that contributes to the overall specificity of polarized trafficking and, perhaps, the formation and maintenance of po- larity itself. This article describes methods that have been designed and adapted specifically for the investigation of SNAREs in epithelial cells. The knowledge of the subcellular localization of a SNARE of interest is essential to understand its function. Un- fortunately, the endogenous expression levels of SNAREs are often low which makes detection challenging. We provide guidelines for determination of the localization of SNAREs by immunofluorescence microscopy including methods for signal amplification, antigen retrieval, and suppression of antibody cross-reactivity. To define which trafficking pathway a SNARE of interest is involved in, one needs to specifically inhibit its function. We provide guidelines for SNARE inhibition by overexpression of the SNARE of interest. An alternative is to introduce inhibitors of SNARE function, such as antibodies or clostridial toxins, into cells. Two methods are presented to make this possible. The first allows the monitoring of effects on trafficking pathways by biochemical assays, and is based on plasma membrane permeabilization using the bacterial toxin streptolysin-O. The second is suitable for single- cell observations and is based on microinjection.