Selection of optimal control samples is crucial in expression profiling tumor samples. To address this issue, we performed microarray expression profiling of control samples routinely used in head and neck squamous cell carcinoma studies: human bronchial and tracheal epithelial cells, squamous cells obtained by laser uvulopalatoplasty and tumor surgical margins. We compared the results using multidimensional scaling and hierarchical clustering versus tumor samples and laryngeal squamous cell carcinoma cell lines. A general observation from our study is that the analyzed cohorts separated according to two dominant factors: "malignancy", which separated controls from malignant samples and "cell culture-microenvironment" which reflected the differences between cultured and non-cultured samples. In conclusion, we advocate the use of cultured epithelial cells as controls for gene expression profiling of cancer cell lines. In contrast, comparisons of gene expression profiles of cancer cell lines versus surgical margin controls should be treated with caution, whereas fresh frozen surgical margins seem to be appropriate for gene expression profiling of tumor samples.