Natural killer T (NKT) cells were first recognized more than two decades ago as a distinct lymphocyte lineage that regulates a broad range of immune responses. The activation of NKT cells paradoxically can lead to either suppression or stimulation of immune responses, but despite this uncertainty, many investigators are hopeful that immune therapies can be developed based on NKT cell modulation. To date, the biology of NKT cells is not well characterized and details of their development have only just started to emerge. It remains unclear how NKT cells migrate from the thymus to the peripheral organs and tissues, and in turn play such diverse roles from one type of immune response to another. Despite this, recent advances in intravital microscopy represent a powerful tool for revealing new insights into NKT cellular dynamics, their patrolling and immunoregulatory functions, which could not have been gained by non-microscopy means. Indeed, imaging has revolutionized the way we visualize with exceptional resolution the cells of the immune system. Instead of seeking a comprehensive review of NKT cell biology, this review attempts to highlight some of the recent studies that use in vivo imaging technologies to address NKT cell responses in a variety of animal models.Immunology and Cell Biology advance online publication, 5 February 2013; doi:10.1038/icb.2013.6.