The ubiquitin-dependent proteolytic pathway plays a major role in selective protein degradation. Ubiquitination of proteins requires the sequential action of the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2), and in some cases ubiquitin-protein ligases (E3s). The oncogenic human papillomavirus (HPV) types 16 and 18 utilize this cellular proteolytic system to target the tumor suppressor protein p53. The HPV E6 oncoprotein binds to a cellular protein of 100 kd, termed E6-associated protein (E6-AP). The E6-E6-AP complex specifically interacts with p53, resulting in the rapid ubiquitin-dependent degradation of p53. Here we report the purification and identification of the factors necessary for the E6-E6-AP-mediated ubiquitination of p53. The ubiquitination of p53 requires the E1 enzyme and a novel E2 in mammalian cells, while E3 activity is conferred by the E6-E6-AP complex. Furthermore, E6-AP appears to have ubiquitin-protein ligase activity in the absence of E6.