The multitude of natural substrates of the 26S proteasome demonstrates convincingly the diversity and flexibility of the ubiquitin/proteasome system: at the same time, the number of pathways in which ubiquitin-dependent degradation is involved highlights the importance of regulated proteolysis for cellular metabolism. This review has addressed recent advances in our understanding of the principles that govern the recognition and targeting of potential substrates. While the mechanism of ubiquitin activation and conjugation is largely understood, the determination of substrate specificity by ubiquitin protein ligases remains a field of active research. Several conserved degradation signals within substrate proteins have been identified, and it is becoming increasingly clear that these serve as docking sites for specific sets of E3s, which in turn adhere to a number of well-defined strategies for the recognition of these motifs. In particular, RING finger proteins are now emerging as a new and apparently widespread class of ubiquitin ligases. The discovery of more and more E3s will undoubtedly reveal even better the common principles in architecture and mechanisms of this class of enzymes. In contrast to substrate recognition by the ubiquitin conjugation system, the way in which a ubiquitylated protein is delivered to the 26S proteasome is poorly understood. There is no doubt that multiubiquitin chains serve as the principal determinant for recognition by the proteasome, and a number of receptors and candidate targeting factors are known, some of which are associated with the proteasome itself; however, unresolved issues are the significance of the different geometries that alternatively linked multiubiquitin chains can adopt, the role of transport between subcellular compartments, as well as the participation of chaperones in the delivery step. Finally, the analysis of ubiquitin-independent, substrate-specific targeting mechanisms, such as the AZ-dependent degradation of ODC, may provide unexpected answers to questions about protein recognition by the 26S proteasome.