Genetic experiments have established an important role for the ubiquitin-like molecule NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) in the regulation of cell growth, viability and development. It is therefore essential to identify the molecular targets for the pathway. Until recently, the cullin family of proteins was characterized as the only substrates for NEDDylation. However, through either direct biological approaches or the use of proteomics, it is now evident that the NEDD8 proteome is more diverse than thought previously. The present review describes the biological significance of NEDDylation for the novel identified substrates and the emerging evidence for the co-operation between the ubiquitin and NEDD8 pathways to control protein function.