Most lung cancer is attributed to long-term smoking. In order to define chromosomal regions with an accumulation of smoking-related early molecular damage, we applied 15 microsatellite markers at 8 chromosomal regions (2q35-q36, 3p21. 3, 3p14. 2, 3p25, 10q11. 2, 11p14-15, 12p13. 1-p12. 3 and 12q14) in an allelotyping study. We studied samples of 42 patients with primary non-small cell lung cancer (NSCLC)(25 squamous cell carcinomas, 13 adenocarcinomas, 2 large cell and 2 bronchioalveolar carcinomas) to compare the frequency of allelic loss in cancer tissue of smokers with matched bronchial epithelium. As a control group we used 11 samples of non-smokers. In NSCLC we found significantly higher frequencies of loss of heterozygosity (LOH) than in matched tumor free bronchial epithelium (p= 0.007). Most frequently, allelic loss was detected in NSCLC at chromosome 3p [3p25 (46%), 3p21. 3 (45%), 3p14 (40%)], at 2q35 (24%), 12p12 (29%) and 12q14 (13%), but infrequently at 10q11 (7%) and 11p14-15 (5%). In corresponding histological normal bronchial epithelium, the highest percentage of LOH was found at chromosome 3p [3p21 (17%), 3p25 (12%), 3p14 (9%)] and chromosome 2q (2q35-q36)(17%) and 12p (12p12-p13)(12%). LOH in histologically normal bronchial epithelium was significantly associated with long-term smoking (p= 0.048), especially at chromosome 12p12 (p= 0.018). Our results demonstrate two further deletion hot spots at the chromosomal region 2q35-q36 and 12p12-p13 in tumor tissue of NSCLC and matched histological normal bronchial epithelium of long-term smokers, reflecting a phenomenon referred to as ‘field cancerization’. These chromosomal regions represent interesting loci for potential NSCLC associated tumor suppressor genes and could be useful as screening markers for molecular risk assessment of smokers.