Gonadotrophin-releasing hormone (GnRH) is a hypothalamic peptide that acts via G(q/11)-coupled 7TM receptors on pituitary gonadotrophs and mediates the central control of reproduction. Recent evidence also indicates that GnRH can affect numerous tissues, but the molecular mechanisms of GnRH receptor stimulation are cell type-specific. Extracellular signal-regulated kinase (ERK) 1 and 2 are key regulators of GnRH function in several cell types, but they also integrate signals from a wide variety of other stimuli. This leads to the obvious question of how specific cellular responses to ERK activation occur, and it is now clear that this is, in part, achieved through strict spatiotemporal control of ERK activity. This means that, in order to infer the function of ERK regulation accurately, multiple readouts for ERK activity, localisation and downstream consequences (e.g. transcriptional activation or cell growth) must be compared simultaneously. Here, we describe some of our findings in the investigation of GnRH signalling to ERK, with particular emphasis on novel, high-content microscopy methods for studying ERK regulation.