Objective: Midkine (MK), a new member of the heparin-binding growth factor family, has been found recently to have a high expression level in many tumor specimens including lung carcinoma. Estrogens may be involved in lung carcinogenesis, and estrogen receptors, mainly estrogen receptor-β (ER-β), are present and functional in normal lung and tumor cell lines and tissues. In addition, estrogens and growth factors may promote the progression of human non-small cell lung cancer (NSCLC). Previously, we have immunohistochemically demonstrated that MK and ER-β proteins were overexpressed in NSCLC and their expression levels were both significantly negatively correlated with the pathological classification. The purpose of this study was to further verify their expression and its correlation with NSCLC.Methods: Taking NSCLC tissues and their corresponding paraneoplastic and normal lung as research objects, we further examined the expression of MK and ER-β by meas of RT-PCR, in situ hybridization and West blot analyses at the levels of messenger RNA (mRNA) and protein, respectively.Results: The increased MK and ER-β mRNA expression was found in NSCLC by RT-PCR and in situ hybridization analyses. Furthermore, West blot analysis also displayed increased expression of MK and ER-β proteins in NSCLC. Finally, their correlation analysis at the levels of mRNA and protein expression in NSCLC demonstrated that MK protein level was significantly correlated to estrogen receptor-β (P<0.01, r_s=0.535); in spite of their correlation at the mRNA level, there was no remarkable difference between MK and ER-β (P>0.05, r_s=0.178).Conclusion: All these results in the present study confirmed that MK and ER-β were overexpressed in human NSCLC.