The receptors for hepatocyte growth factor (HGFR), epidermal growth factor (EGFR) and insulin (INSR) are transmembrane receptor tyrosine kinases that activate various downstream signalling pathways from their phosphotyrosine residues upon ligand binding. Of these, the PI3K/Akt pathway is responsible for antiapoptotic/survival effects, but in the case of the INSR, PI3K is also thought to mediate the metabolic effects of insulin. It was earlier found that in HepG2 hepatic carcinoma cells the p85 subunit of PI3K associates and co-precipitates with Gab1, EGFR and IRS1 upon HGF, EGF and INS stimulation, which are the markers of global activation along these respective pathways. However, the p110beta subunit of PI3K only co-precipitates with IRS1 upon INS stimulation, but not upon the activation of the other two pathways. To examine the specificity of PI3K p110beta in mediating insulin stimulated responses, we have optimized multicolor immunofluorescent labeling and performed confocal microscopic colocalization experiments to trace the selective association of this subunit with INSR and the possible differential localization of HGFR, INSR and PI3K p110beta in GM1 positive lipid microdomains. Our results indicate that while both HGF and INS treatments recruit their respective receptors into lipid rafts, PI3K p110beta is only recruited to the rafts by INS and its association with INSR is also significantly increased after a 2 min treatment with insulin. Given that PI3K p110beta contains a unique phosphorylation site not present in p110alpha, we suspect that the p110beta may be responsible specifically for the metabolic effects of insulin, while p110alpha could be mediating in the antiapoptotic pathways.