The exon 16-encoded juxtamembrane (JM) domain of human insulin receptor (hIR) harbors the NPEY motif which couples the insulin-activated hIR kinase to downstream signal transduction molecules. We sought to determine if signal transduction requires the entire exon 16-encoded 22-amino acid JM domain. Transfected CHO cells were generated stably expressing either the wild-type hIR (hIR-WT) or two mutant hIRs (hIRΔEx16 in which the JM domain was deleted, and hIRrosJM in which the deleted segment was replaced by the corresponding domain of v-ros protein). The mutant hIRΔEx16 and hIRrosJM exhibited similar insulin-binding as the hIRWT. Insulin internalization and insulin dose-response experiments toward activation of downstream signal transduction molecules demonstrated that: i) the presence of intact hIR-JM domain which harbors the NPEY motif is essential for Shc phosphorylation but not for IRS-1 phosphorylation; ii) insulin signal transduction can occur independent of the JM domain of hIR and without participation of the NPEY motif; iii) engagement of this putative alternative downstream signal transduction is Shc independent and is dependent on insulin concentration; and iv) insulin internalization does not necessarily require the hIR specific aa sequence of the JM domain which can be partially substituted by the JM domain of the v-ros tyrosine kinase.