We used oligonucleotide microarrays to investigate gene expression changes associated with multi‐step human papillomavirus type 16 (HPV16)‐mediated carcinogenesis in vitro. Gene expression profiles in 4 early passage HPV16‐immortalized human keratinocyte (HKc) lines derived from different donors were compared with their corresponding 4 late‐passage, differentiation‐resistant cell lines, and to 4 pools of normal HKc, each composed of 3 individual HKc strains, on Agilent 22 k human oligonucleotide microarrays. The resulting data were analyzed using a modified T‐test coded in R to obtain lists of differentially expressed genes. Gene expression changes identified in this model system were then compared with gene expression changes described in published studies of cervical intraepithelial neoplasia (CIN) and cervical cancer. Common genes in these lists were further studied by cluster analysis. Genes whose expression changed in the same direction as in CIN or cervical cancer (concordant) at late stages of HPV16‐mediated transformation in vitro formed one major cluster, while those that changed in the opposite direction (discordant) formed a second major cluster. Further annotation found that many discordant expression changes involved gene products with an extracellular localization. Two novel genes were selected for further study: overexpression of SIX1 and GDF15, observed during in vitro progression in our model system, was confirmed in tissue arrays of cervical cancer. These microarray‐based studies show that our in vitro model system reflects many cellular and molecular alterations characteristic of cervical cancer, and identified SIX1 and GDF15 as 2 novel potential biomarkers of cervical cancer progression. © 2008 Wiley‐Liss, Inc.