The growth arrest DNA-damage-inducible protein 45 (GADD45) can serve as a key coordinator of the stress response by regulating cell cycle progression, genomic stability, DNA repair, and other stress-related responses. Although deregulation of GADD45 expression has been reported in several types of human tumors, its role in lung cancer is still unknown. DNA hypermethylation of promoter CpG islands is known to be a major mechanism for epigenetic inactivation of tumor suppressor genes. We investigated the methylation status of GADD45 family genes (GADD45A, B, and G) in 139 patients with non-small cell lung cancer (NSCLC) using methylation-specific PCR (MSP) and correlated the results with clinicopathologic features of the patients. Methylation frequencies in tumors were 1.4% for GADD45A, 7.2% for GADD45B, and 31.6% for GADD45G. RT-PCR and MSP analysis showed that promoter methylation of the GADD45G gene resulted in down-regulation of its mRNA expression. GADD45G methylation was significantly more frequent in female patients than male patients (P = 0.035). This finding suggests that methylation-associated down-regulation of the GADD45G gene may be involved in lung tumorigenesis.