We recently characterized the association of the 44-kDa mitogen-activated protein kinase, also known as extracellular-regulated kinase 1 (ERK1), with the 90-kDa ribosomal S6 kinase (pp90rsk), one of its putative substrates in intact PC12 cells. Using antibodies to ERK1 that precipitate a functional ERK1.pp90rsk phosphoprotein complex, we demonstrate here the regulation of both kinases by various stimuli. In mouse fibroblasts expressing human insulin receptors, insulin and vanadate swiftly stimulated ERK1 activity within 5 min. While the hormonal effect was short-lived, vanadate led to a first peak followed by a progressively increasing second phase. In PC12 cells, epidermal growth factor, which is a growth promoting factor, provokes a rapid but evanescent activation of ERK1. In contrast, nerve growth factor (NGF), which acts as a neuronal differentiation factor for PC12 cells, induced a swift monophasic response followed by a sustained second phase. This strikingly different pattern of ERK1 stimulation by NGF and epidermal growth factor was associated to a contrasting effect on ERK1 cellular translocation. Thus, NGF induced a nuclear translocation of ERK1, while epidermal growth factor was without noticeable effect on ERK1 localization. In both cell systems all effectors tested stimulated ERK1 phosphorylation on both threonine and tyrosine residues in an 1:1 ratio. During ERK1 inactivation, phosphothreonine and phosphotyrosine were dephosphorylated in a similar fashion. Concurrent with ERK1 activation was the de novo appearance of phosphothreonine and an increase in phosphoserine on pp90rsk. The pp90rsk phosphothreonine content paralleled the ERK1 activity more closely than the phosphoserine level. These results provide compelling evidence that in fibroblasts and PC12 cells ERK1 plays a direct role in the phosphorylation of pp90rsk and that pp90rsk represents a physiologically relevant substrate of extracellular-regulated kinases. Finally, we would like to suggest that the differentiating action of NGF in PC12 cells might be due, at least in part, to the conjunction of its sustained and robust stimulation of ERK1 and pp90rsk, and of its induction of ERK1 nuclear translocation.