High continuous GH levels in vivo produce desensitization of the Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway of GH signaling in the liver. To evaluate the mechanisms involved in this desensitization, transgenic mice overexpressing bovine GH were used. In these animals, GH receptor and membrane-associated JAK2 kinase are increased 4.5- and 6-fold, respectively. However, JAK2. STAT5a and -5b do not become tyrosine phosphorylated in response to GH stimulus, nor are these STAT proteins recruited to membranes, suggesting that they cannot bind to the receptor. The content of the suppressor cytokine-inducible src homology 2 (SH2)-containing protein (CIS), both total and membrane-associated, is markedly increased in the liver of GH transgenic mice. This could account for the inhibition of STAT5 activation, because CIS competes with STAT5 for GH receptor docking sites. Existence of an alternative mechanism of negative regulation of this signaling pathway by chronically elevated GH levels is suggested by the low level of JAK2 phosphorylation that transgenic mice exhibit. Whereas total SH2-containing phosphatase 2 (SHP-2) content is the same in both kinds of mice, membrane-associated SHP-2 protein levels increase 4.5-fold in GH transgenic animals. This could explain the dramatic inhibition of JAK2 phosphotyrosine level, thus contributing to the suppression of GH signaling observed in these transgenic mice.