Increased activity of the ubiquitin-proteasome system (UPS), resulting in an imbalance between protein degradation and protein synthesis, is thought to be central to the loss of mass of skeletal muscle undergoing atrophy. Both animal and human studies consistently demonstrate that, as part of the muscle atrophy programme, expression of the E3 ubiquitin-ligase enzymes MAFbx and MuRF1 (gene products of so-called ‘atrogenes’) is significantly increased. Further, MAFbx and MuRF1 knockout mice preserve muscle mass and fibre-size when subject to denervation or disuse atrophy, indicating they are required for atrophy to occur. This upregulation of both MAFbx and MuRF1 is likely to result in increased ubiquitylation of specific muscle proteins, and presumably, their subsequent degradation by the 26S proteasome complex. Recently, a new ‘atrogene’ designated Znf216 has been identified [Hishiya et al., 2006]. The ZNF216 protein possesses Znf-A20 and Znf-AN1 domains at its N- and C-termini, respectively, both of which can be linked to the UPS. In order to further understand its role in muscle homeostasis, we have initiated an in vitro functional characterisation of the rat ZNF216 protein. The coding region of Znf216 was cloned from mRNA isolated from rat skeletal muscle, which enabled recombinant expression (in E. coli) of a soluble glutathione S-transferase tagged protein (GST-rZNF216); milligram quantities of GST-rZNF216 were successfully purified using glutathione-Sepharose affinity chromatography. Initial work indicated that rZNF216 can self-associate and form homo-oligomers. Despite the presence of the Znf-A20 domain previously found in some deubiquitylating (DUB) enzymes, rZNF216 displayed no apparent DUB activity in vitro. However, rZNF216 clearly interacted with immobilised monoubiquitin (ubiquitin-Sepharose) in addition to unanchored K48- and K63-linked polyubiquitin chains, in a linkage-independent manner. Furthermore, affinity chromatogaphy experiments have shown that rZNF216 can efficiently precipitate ubiquitylated proteins from skeletal muscle extracts, whilst at the same time, revealing other proteins which may interact directly with rZNF216. Since ZNF216 is thought to interact with both the 26S proteasome and ubiquitylated proteins, we propose that its role may be to act downstream of MAFbx/MuRF1 in the atrophy programme, delivering ubiquitylated substrates of MAFbx/MuRF1 to the proteasome. Thus, identification of ZNF216-interacting proteins from skeletal muscle could provide further insights into the molecular mechanisms that regulate the muscle atrophy programme, and may also help uncover the elusive in vivo substrates of MAFbx and/or MuRF1.