The growth hormone receptor (GHR) is a cell surface receptor that mediates the somatogenic and metabolic effects of the growth hormone (GH). GHR signaling is transduced via the receptor-associated cytoplasmic tyrosine kinase called Janus protein kinase 2 (JAK2). The major intracellular signaling systems activated by JAK2 in response to GH include the signal transducer and activator of transcription (STAT) 5 and extracellular signal-regulated kinase (ERK)-1 and -2 pathways. In this report, we investigate the role of cholesterol-rich plasma membrane microdomains (caveolae and lipid rafts) in GH signaling. By subcellular fractionation of the GH-responsive 3T3-F442A murine preadipocyte, we found dramatic enrichment (6.7-fold) of plasma membrane GHR in the caveolae membranes (CM). JAK2 was also represented in the CM fraction, but was less enriched (2.5-fold) than GHR. ERK1/2 and the important ERK pathway upstream small adaptor protein, Grb2 (growth factor receptor-bound protein 2), were also enriched in caveolae (2.3- and 8.3-fold, respectively), but STATS was barely detected in the same fraction. Correspondingly, GH-induced tyrosine-phosphorylated GHR, JAK2, and ERK1/2 were highly represented in the CM fraction, whereas tyrosine-phosphorylated STATS was enriched in the non-membranous fraction of the post-nuclear supernatant. Additionally, GH induced further accumulation of GHR, Grb2, and SHC proteins in the CM fraction. Interestingly, treatment of the cells with the caveolae-disrupting agent, methyl-β-cyclodextrin (mβCD), selectively inhibited GH-induced ERK1/2 activation but not STAT5 hosphorylation; repletion of cholesterol in mβCD-treated cells restored GH-induced ERK activation. Comparison of 3T3-F442A cells with the GHR-expressing human IM-9 lymphoblasts revealed similar enrichment of GHR in the lipid raft fraction of IM-9 as in the CM fraction of 3T3-F442A, but there were dramatic differences in the ERKs and Grb2. The IM-9 cell, in which ERKs are not activated by GH, displayed no enrichment of ERKs and Grb2 in the lipid raft fraction. Our results suggest that localization of GHRs in the CM fraction of the plasma membrane plays important roles in signaling.