The biological effects of growth hormone (GH) are initiated by its binding to the GH receptor (GHR) followed by association and activation of the tyrosine kinase JAK2. Here we report that GH can stimulate an increase in intracellular free Ca2+ concentration ([Ca2+]i) in cells expressing wild-type GHRs and receptor mutants lacking up to 132 amino acids of the C terminus, whereas GHRs lacking a further 52 amino acids in the C terminus are unable to induce Ca2+ signaling. The GH-induced rise in [Ca2+]i was dependent upon extracellular Ca2+ and the response consisted of GH-induced Ca2+ oscillations of varying frequency and amplitude. GH-induced transcription of the serine protease inhibitor 2.1 gene required the same C-terminal 52-amino acid domain of the receptor as for Ca2+ signaling. Mutation of the four proline residues in the conserved box 1 region of the GHR, which is responsible for binding and activation of JAK2 kinase, completely abolished GH-induced gene transcription but did not affect the GH-induced rise in [Ca2+]i. The Ca2+ channel blocker verapamil prevented GH-induced Ca2+ signaling as well as GH-induced gene transcription in cells expressing endogenous GHRs. These findings indicate that the GHR can initiate two independent signaling pathways, one requiring the box 1 region and the other requiring the region between amino acids 454 and 506, and suggest that both of these pathways are required for GH-induced gene transcription.