Understanding the dynamic control of hormonal and metabolic homeostasis requires a description of the input, secretory and output mechanisms that underlie the life cycle of hormone pulses. Whilst input stimuli and hormone output have been measured, in vivo measurements of the blood microcirculation during a secretory pulse, and signalling at the cell and population level in the intact organ have not. To achieve this long-standing question, we recently developed a strategy to image in real-time the life cycle of hormone pulses in the pituitary gland by using a new imaging approach utilising long range objectives (Lafont et al. PNAS 2010 107 44654470. It enabled us to measure local blood flow, oxygen partial pressure and cell activity at single-cell resolution in mouse pituitary glands in situ. When secretagogue (GHRH) distribution was modelled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. Moreover, we observed a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Hence, GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context, involving a functional association between the GH cell network activity and fluid microcirculation. These new cellular in vivo imaging approaches will allow the future investigation of how the pituitary microenvironment influences different cell systems, not only during periods of normal physiological demand, but also when the endocrine tissue and microvasculature are altered (e.g. tumours).