Chronic inflammation has been implicated as a cofactor in the development of several human cancers. Interleukin-1beta is a key pro-inflammatory cytokine. We have previously shown associations between polymorphisms at position -511 and -31 in the promoter of the IL1B gene and risk of non-small cell lung cancer. In this study we investigated the functional role of the -31 T/C SNP in the regulation of the IL1B gene. The -31 T/C polymorphism is located in the core promoter and may affect the binding of transcription factors and thereby promoter activity. We therefore established a promoter reporter assay to explore the impact of the promoter variants on the expression of the gene. In the present study, we show that expression from the T-variant of the promoter was higher (p<0.001) than from the C-variant, in A549 cells. Electrophoretic mobility shift assays revealed differential binding of proteins to a promoter fragment containing the SNP. Interestingly, a highly specific complex formed on the C-variant that was not present on the T-variant. Supershift experiments implicated binding of both the CCAAT-enhancer binding protein beta (C/EBPbeta, also called NF-IL6) transcription factor and the TATA-box binding protein (TBP) to the SNP region. Due to the high frequency of this SNP, differences in IL1B gene expression caused by the variants may have significant biological impact in the population.