UBIQUITINATION Of proteins involves the concerted action of the El ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-protein ligases(1-3). It has been proposed that E3s function as ’docking proteins’, specifically binding substrate proteins and specific E2s, and that ubiquitin is then transferred directly from E2s to substrates(1-5). We show here that formation of a ubiquitin thioester on E6-AP, an E3 involved in the human papillomavirus EB-induced ubiquitination of p53 (refs 6-10), is an intermediate step in E6-AP-dependent ubiquitination. The order of ubiquitin transfer is from E1 to E2, from E2 to E6-AP, and finally from E6-AP to a substrate. This cascade of ubiquitin thioester complexes suggests that E3s have a defined enzymatic activity and do not function simply as docking proteins. The cysteine residue of E6-AP responsible for ubiquitin thioester formation was mapped to a region that is highly conserved among several proteins of unknown function, suggesting that these proteins share the ability to form thioesters with ubiquitin.