Defining the function of all proteins in an organism is one of the major objectives for biology in the coming decades. Here, we assess approaches used to determine subcellular protein location and discuss the relationship between protein location and function. It is important to recognize that targeting, accumulation, and the site of function are not necessarily interchangeable terms with respect to defining the location of a protein. Some proteins have tightly defined locations, whereas others have low specificity targeting and complex accumulation patterns. Location may be essential for function in some cases, but it may be much less important for other proteins. There is no single approach that can be considered entirely adequate for defining the in vivo location of all proteins. By combining approaches that assess targeting and accumulation of proteins, more confidence can be gained about localization. The strengths and weaknesses of different localization technologies are summarized, and some guidelines for performing combined targeting and accumulation assays are outlined.