Mitogenic signal-transduction pathways have not been well defined in pancreatic β-cells. In the glucose-sensitive rat β-cell line, INS-1, glucose (6-18 mM) increased INS-1 cell proliferation (> 20-fold at 15 mM glucose). Rat growth hormone (rGH) also induced INS-1 cell proliferation, but this was glucose-dependent in the physiologically relevant concentration range (6-18 mM glucose). The combination of rGH (10 nM) and glucose (15 mM) was synergistic, maximally increasing INS-1 cell proliferation by > 50-fold. Moreover, glucose-dependent rGH-induced INS-1 cell proliferation was increased further by addition of insulin-like growth factor 1 (IGF-1; 10 nM) to > 90-fold at 12 mM glucose. Glucose metabolism and phosphatidylinositol-3ʹ-kinase (PI3ʹK) activation were necessary for both glucose- and rGH-stimulated INS-1 cell proliferation. Glucose (> 3 mM) independently increased tyrosine-phosphorylation-mediated recruitment of growth-factor-bound protein 2 (Grb2)/murine sons of sevenless-1 protein (mSOS) and PI3ʹK to insulin receptor substrate (IRS)-1 and IRS-2, as well as SH2-containing protein (Shc) association with Grb2/mSOS and downstream activation of mitogen-activated protein kinase and 70 kDa S6 kinase. Glucose-induced IRS- and Shc-mediated signal transduction was enhanced further by the addition of IGF-1, but not rGH. In contrast, rGH was able to activate Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) signal transduction at glucose concentrations above 3 mM, but neither glucose independently, nor glucose with added IGF-1, were able to activate the JAK2/STAT5 signalling pathway. Thus rGH-mediated proliferation of β-cells is directly via the JAK2/STAT5 pathway without engaging the Shc or IRS signal-transduction pathways, although activation of PI3ʹK may play an important permissive role in the glucose-dependent aspect of rGH-induced β-cell mitogensis. The additive effect of rGH and IGF-1 on glucose-dependent β-cell proliferation is therefore reflective of rGH and IGF-1 activating distinctly different mitogenic signalling pathways in β-cells with minimal crosstalk between them.