The receptor that mediates the increase in glucose transport (GT) in response to β-adrenoceptor (β-AR) agonists was characterized in the rat skeletal muscle cell line L6, using the 2-deoxy-[3H]-D-glucose assay.The β3-AR agonist BRL37344 (pEC50=6.89±0.21), the β-AR agonist isoprenaline (pEC50=8.99±0.24) and the β2-AR agonist zinterol (pEC50=9.74±0.15) increased GT as did insulin (pEC50=6.93±0.15). The highly selective β3-AR agonist CL316243 only weakly stimulated GT.The pKB values calculated from the shift of the pEC50 values of the agonists in the presence of the β1-AR selective antagonist CGP 20712A or the β3-AR selective antagonist SR 59230A were not indicative of activation of β1- or β3-ARs. Only (−)-propranolol and the β2-AR selective antagonist ICI 118551 caused marked rightward shifts of CR curves to isoprenaline (pKB=10.2±0.2 and 9.6±0.3), zinterol (pKB=9.0±0.1 and 9.4±0.3) and BRL 37344 (pKB=9.4±0.3 and 8.4±.2), indicating participation of β2-ARs.The pharmacological analysis was supported by reverse transcription and polymerase chain reaction analysis of L6 mRNA, which showed high levels of expression of β2-AR but not β1- or β3-AR in these cells.Forskolin and dibutyryl cyclic AMP produced negligible increases in GT while the phosphatidylinositol-3 kinase inhibitor, wortmannin, significantly decreased both insulin- and zinterol-stimulated GT, suggesting a possible interaction between the insulin and β2-AR pathways.This study demonstrates that β2-ARs mediate the increase in GT in L6 cells to β-AR agonists, including the β3-AR selective agonist BRL 37344. This effect does not appear to be directly related to increases in cyclic AMP but requires P13K.