EGFR (ErbB1) and ErbB2 receptors stimulate several intracellular signaling pathways in non-small-cell lung cancer (NSCLC). Adenocarcinomas (AC) and squamous cell carcinomas (SCC) are NSCLC subtypes with distinct clinico-pathological features, and responses to ErbB-targeted inhibitors treatment. To evaluate the causes of these differences, tissue microarrays with samples from NSCLC patients (189 AC and 56 SCC) were used to study EGFR and ErbB2 expression and phospho-activation of ERK1/2, AKT, STAT3 and SRC ErbB-mediators by immunohistochemistry and Western blot, and EGFR and ErbB2 gene amplification by FISH. EGFR expression was higher in SCC than in AC (P<0.001), while ErbB2 showed similar low levels. Phosphorylated (p) ERK, pAKT, pSTAT3 and pSRC levels were prevalent in AC (P< or =0.002). EGFR levels and signaling mediators activation were differentially associated with each of the pathologies. Whereas in AC the expression and amplification of EGFR were linked to AKT activation (P< or =0.050), in SCC its expression was correlated with pSTAT3 (P=0.024). In addition, pSTAT3 was correlated with pERK and pAKT only in AC (P< or =0.045). Biomarker levels were also differentially associated with the clinico-pathologic variables. In AC, EGFR and pSRC increasing scores correlated with female sex and the smoking habit (P< or =0.008), while ErbB2 amplification increased with advanced age and tumor stage (P< or =0.047), and pERK1/2 and pSTAT3 levels correlated with early tumor stage (P< or =0.045). In SCC, EGFR amplification was stronger in younger patients (P=0.013), pERK1/2 in the older ones (P=0.050), and pSTAT3 amplification was stronger in women (P=0.001). These data support that AC and SCC lung tumors are distinct entities at the molecular level, and that their signaling status in combination with their clinico-pathologic variables may be considered for differential targeted therapies.