We have previously established that the eleven cytosolic peptides phosphorylated in response to acute glucagon challenge in isolated rat hepatocytes undergo rapid dephosphorylation following transfer to medium free of 32PO4(3-). This dephosphorylation, far from being a simple process, is complex and asynchronous. This novel finding of asynchrony raises the question of whether, by analogy to glucagon, protein dephosphorylation is asynchronous during the recovery phase from acute challenge with noradrenaline, vasopressin or angiotensin II. One-dimensional SDS-PAGE of hepatocyte extracts indicates that noradrenaline stimulates the phosphorylation of ten cytosolic peptides, whereas vasopressin and angiotensin II stimulate the phosphorylation of six cytosolic peptides. Transfer of the hormone-challenged hepatocytes to medium devoid of 32PO4(3-) and hormone led to the rapid net dephosphorylation of the 32P-labelled phosphopeptides, albeit at different rates. In all instances, the most rapidly dephosphorylated phosphopeptide was glycogen phosphorylase. Statistical analysis indicates that during recovery from noradrenaline challenge three distinct groups of phosphopeptides can be delineated on the basis of their rates of dephosphorylation. Despite the fact that vasopressin and angiotensin II stimulate the phosphorylation of the same sub-set of phosphopeptides, there were differences in the rates of dephosphorylation of these phosphopeptides during the recovery phase from acute hormonal challenge.