Polyphenol oxidases (PPOs) have been implicated in plant defence reactions. From an applied point of view, high PPO activity is associated with browning / darkening of fresh and processed food. Owing to its complex genome and economic importance, wheat (Triticum aestivum L.) represents an interesting system to advance our understanding of plant PPO function. We have previously shown that wheat PPOs are organised in a multigene family, consisting of two distinct phylogenetic clusters with three members each. In this study, we demonstrate that members of one cluster are not expressed in developing kernels or senescing flag leaves. Transcriptional regulation of one major gene in the other cluster largely controls PPO levels in these tissues, at least in the wheat varieties used for this study. Our data further indicate that the product of this gene is present as a latent enzyme during early kernel development, and that the latent enzyme is activated during later developmental phases. Enzyme activation can be achieved in vitro by limited tryptic digestion, but our data do not indicate activation by a proteolytic mechanism in vivo. Together, results presented in this study provide important insights into the regulation of wheat PPO function.