BACKGROUND: RNA interference represents a powerful tool with which to achieve suppression of specific target mRNA. Real-time rtPCR is a useful technique for assessing levels of mRNA expression. Critically, for real-time rtPCR to yield meaningful results, it is necessary to normalise expression of the gene of interest to stably expressed endogenous control genes. OBJECTIVES: The study involved establishing expression profiles for 11 housekeeping genes in human epidermal keratinocyte cell lines determining their relative stability. Furthermore, the effect of the presence of shRNA on these expression profiles has been established. METHODS: Keratinocytes were transfected using lipid-based transfection or AMAXA nucleofection. Real-time rtPCR was used to establish RNA expression levels. Data analysis was carried out using geNORM. RESULTS: When using HaCaT or adult NHEK cells any combination of 8 of the housekeeping genes would be appropriate for normalisation. In contrast, with juvenile NHEK cells only 4 of the housekeeping genes were found to be sufficiently stable to be deemed appropriate for normalisation of expression data. Furthermore data demonstrated that the expression of housekeeping genes may sometimes be affected by the induction of the RNAi pathway. CONCLUSIONS: The data obtained highlight the importance of characterising housekeeping gene expression profiles in each specific cell type prior to choosing a set of housekeeping genes for expression studies. The results from this study are applicable to researchers working with human epidermal keratinocytes and the experimental approach is more broadly applicable to any researcher carrying out real-time rtPCR.