Gap junctions are mediated by intercellular channels that connect adjacent cells and are composed of Connexin (Cx) proteins. A member of the Cx family, Cx26 is considered a potential tumor suppressor in several cancers. The expression of Cx26 gene and its methylation status in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) were investigated. Six-wk-old male Wistar rats were given 2,000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until they were sacrificed at 25 wk. A total of nine lung adenocarcinomas were obtained and total RNA was extracted to assess expression by real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR). Five out of nine adenocarcinomas showed reduced expression compared with normal lung tissue. We next performed a bisulfite sequence analysis to measure the methylation status of the 5' upstream region of the Cx26 gene in two normal lung tissues and five lung adenocarcinomas that showed reduced expression of Cx26. All five adenocarcinomas were highly methylated in the 5' upstream region, while the two normal lung tissues were unmethylated. This suggests that aberrant methylation of the Cx26 gene may be involved in the development of lung adenocarcinomas induced by BHP in rats.