The present work is designed to study the expression level of both IRS-1 and IRS-2 in primary mouse hepatocytes (PMH), and investigate their kinetics of phosphorylation in response to addition of variable doses of insulin. The present study also involves determination of subcellular localization of both IRS-1 and IRS-2 in PMH using confocal microscopy. Downregulation of IRS-2 in PMH using siRNA against IRS-2 delivered into hepatocytes by means of adenovirus infection was done. The effect of this downregulation on the expression of IRS-1 protein and the phosphorylation of both IRS-1 and IRS-2 was studied.Results reveal higher expression level of IRS-2 than IRS-1 in PMH. On addition of insulin IRS-2 shows faster kinetics of phosphorylation than IRS-1 but the degree of phosphorylation of IRS-1 is higher than IRS-2. Results also show that IRS-2 is mainly localized in cell membrane while IRS-1 is homogenously distributed all over the cytoplasm and in nuclei of PMH. Addition of insulin induces nuclear translocation of IRS-1 and to lesser extent IRS-2. Adenoviral infection of PMH may cause endoplasmic reticulum and cellular stress and could affect the expression of IRS proteins in these cells.