Tumor Suppressor in Lung Cancer-1 (TSLC1) expression is repressed in many different cancers. Hypermethylation of six CpG sites upstream of the TSLC1 coding region is correlated with TSLC1 repression. However, the functional elements of the TSLC1 promoter have not been examined. In this study, the transcription start site was identified as being 67 or 62 bp upstream of the translational start site by primer extension and bioinformatics analysis of expressed sequence tags (ESTs), respectively. Two promoter regions that regulate TSLC1 expression were identified. One, a small region of 170 bp, including 107 bp upstream of the transcription initiation site, is sufficient to generate maximal TSLC1 promoter activity. This minimal promoter does not contain the CpG sites that are hypermethylated in repressed TSLC1 promoters. Two, the maximal activity of the TSLC1 promoter is dampened by an inhibitory region that resides upstream of the minimal promoter.