ERK1b is an alternatively spliced form of ERK1, containing a 26-amino acid insertion between residues 340 and 341 of ERK1. Although under most circumstances the kinetics of ERK1b activation are similar to that of ERK1 and ERK2, we have previously found several conditions under which the activation of ERK1b by extracellular stimuli differs from that of other ERKs. We studied the molecular mechanisms that cause this differential regulation of ERK1b and found that ERK1b is altered in its ability to interact with MEK1 and this influenced its subcellular localization but not its kinetics of activation. ERK1b had a decreased ability to phosphorylate Elk1, but this did not change much the transcriptional activity of the latter. Importantly, the interaction of ERK1b with PTP-SL, which can act as a MAPK phosphatase, shortly after mitogenic stimulation, was significantly affected as well. Using mutants of ERK1b we found that the differential interaction of ERK1b with the three effectors is caused by the site of insertion that abrogates the cytosolic retention sequence/common docking motif of ERKs, and is not dependent on the actual sequence of the insert. Prolonged epidermal growth factor stimulation of Rat1 cells resulted in a differential inactivation and not activation of ERK1b as compared with ERK1 and ERK2. The reduced sensitivity to phosphatases without major differences in the kinetics of activation or activation of substrates, suggests that ERK1b plays a role in the transmission of extracellular signals under conditions of persistent stimulation, where ERK1b and MAPK phosphatases are induced, and the activity of ERK1 and ERK2 is suppressed.