The ARF tumour suppressor gene encodes a small highly basic protein whose known functions are largely determined by the amino acids encoded within the first exon. In mammals, the protein incorporates additional residues specified by an alternative reading frame in the second exon of INK4a, but this arrangement does not apply to the chicken homologue. In exploring the intracellular localization of chicken p7(ARF), we found that while the FLAG- and HA-tagged versions localize in the nucleolus, in line with mammalian ARF, the GFP-tagged version is excluded from the nucleolus. Here we show that irrespective of the source or composition of the ARF fusion proteins, versions that accumulate in the nucleolus share the ability to bind to nucleophosmin (NPM). Depletion of NPM with siRNA results in the re-location and destabilization of nucleolar forms of ARF but has little effect on the location or stability of a nucleoplasmic form of ARF. Importantly, knockdown of endogenous NPM does not impair the ability of ARF to bind to MDM2 and stabilize p53. These findings support the view that nucleolar localization determines the stability of ARF but not its primary function.