The yeast two-hybrid system has provided a convenient means to both screen for proteins that interact with a protein of interest and to characterise the known interaction between two proteins. Several groups with an interest in the molecular mechanisms that underlie discrete steps along trafficking pathways have exploited the yeast two-hybrid system. Here, we provide a brief background to the technology, attempt to point out some of the pitfalls and benefits of the different systems that can be employed, and mention some of the areas (within the trafficking field) where yeast two-hybrid interaction assays have been particularly informative.