Growth hormone treatment (GH) decreases adipose tissue sensitivity to insulin. However, the exact molecular mechanism(s) involved remains unclear. In the present study, we have evaluated the chronic effects of GH on adipose tissue explants cultured in a defined media. The objective was to determine the effects of GH treatment for 24 and 48 hours on the early steps of the insulin signal transduction, including IRS-3. The 24-hour culture media contained no hormones or 100 ng/ml GH. The 48-hour culture media contained insulin and dexamethasone supplemented with or without 100 ng/ml of GH. Results demonstrated a reduction in the cellular concentration of IRS-1 by around 30% when adipose tissue was chronically treated with growth hormone for either 24 or 48 hours. IRS-3 protein levels were also decreased by 15% after the 24-hour treatment, and by 27% after culture with GH for 48 hours in the presence of insulin and dexamethasone. PI 3-kinase concentrations were also reduced by GH in both experiments by around 25%. At the end of the 24-hour culture with GH adipose explants were stimulated with insulin in a short-term incubation, after which phosphorylation and association of the IRSs with PI 3-kinase were evaluated. After the insulin stimulus, the association of PI 3-kinase with IRS-1 and IRS-3 were decreased in explants chronically cultured with GH by 44 and 28%, respectively. After this short-term insulin stimulus, the IRS-3 phosphorylation was also lowered in GH-treated explants. The results with chronic cultures of adipose presented here are consistent with similar changes in IRS-1 and IRS-2 concentration and phosphorylation observed for liver and muscle after long-term (3-5 days) in vivo treatment with GH. The data suggest that chronic GH treatment alters the early steps of the insulin signal transduction pathway, and may explain the changes in adipose tissue sensitivity to insulin.