Dendritic cells (DC) are central regulators of immune responses. Their functional characterization has, thus far, mainly relied on the analysis of ex vivo isolated cells or immunohistology, which provides information in a static manner. While these approaches have enabled an excellent understanding of the role of DC in antigen uptake, processing and presentation, there has been a clear need to investigate the behaviour of DC in the context of intact tissues in real time. This demand has recently been met by the availability of intravital two-photon microscopy, which allows for the visualization of single cells deep within intact organs over time. Thus, during the past few years, exciting new data have been generated as to how DC behave within secondary lymphoid and peripheral tissues both under homoeostatic and inflammatory conditions. Here, we will review what two-photon microscopy studies have taught us about the migration of DC in the interstitial space as well as their interactions with adaptive immune cells.