New methods for the analysis of glucose transporters were used to analyze the molecular mechanisms involved in the insulin-antagonistic effects of growth hormone (GH), which is known as a diabetogenic hormone. The ability of GH to alter the number and mRNA levels of two different glucose transporters in cultured 3T3-F442A adipocytes was investigated using specific antibodies and cDNA probes. At concentrations of GH as low as 0.5 and 5 ng/ml and at incubation times as short as 4 h, GH decreased rates of 2-deoxyglucose uptake in 3T3-F442A adipocytes. 3-O-Methyl-D-glucose uptake was inhibited to an extent similar to that of 2-deoxyglucose uptake (60-80%) after a 24-h incubation with GH (500 ng/ml), indicating that GH inhibits glucose metabolism specifically at the step of glucose transport. To determine whether reduced rates of glucose transport might result from reduced numbers of glucose transporters, whole cell lysates were prepared from GH-treated cells and subjected to immunoblotting using antibodies that identify Glut 1 (HepG2/rat brain) and Glut 4 (muscle/adipose) transporters. GH caused a time- and dose-dependent decrease in the number of Glut 1 transporters in the cell. Northern and slot-blot analyses showed a GH-induced dose-dependent decrease in levels of Glut 1 mRNA. In contrast, levels of Glut 4 transporter and mRNA were unchanged by GH. These data suggest that GH regulates Glut 1 and Glut 4 transporters differentially and that it exerts its inhibitory effect on glucose uptake at least in part by decreasing the synthesis of Glut 1 transporters. These studies provide the first evidence that GH regulates a key gene in metabolic regulation and can interfere with gene expression.