AbstractUbiquitin specific proteases (USPs) regulate the production and recycling of ubiquitin and are thereby critically involved in the control of cell growth, differentiation, and apoptosis. Increasing evidence implicates deregulation of USPs in malignant transformation but there is very little information on the overall and specific activity of USPs in normal and tumor tissues. We have used a chemistry‐based functional proteomics approach to profile the activities of individual USPs in biopsies of human papillomavirus (HPV) carrying cervical carcinoma and adjacent normal tissue. To assess the contribution of HPV proteins, USP activity was also compared in HPV positive and negative cervical carcinoma cell lines and HPV E6/E7 immortalized human keratinocytes. The activity of the C‐terminal hydrolases UCH‐L3 and UCH37 was upregulated in the majority of tumor tissues compared to the adjacent normal tissues. UCH‐L1 activity was lower in a significant proportion of the tumors but to a less extent in advanced tumors. In accordance with the relatively low UCH‐L1 activity in tumor biopsies, UCH‐L1 was detected only in one out of eight cervical carcinoma lines. UCH‐L1, UCH‐L3, USP7, and USP9X activity was upregulated following HPV E6/E7 immortalization of keratinocytes, suggesting a role of these enzymes in growth transformation. © 2006 Wiley‐Liss, Inc.