Hyperparathyroidism 2 (HRPT2) gene mutations underlie hereditary and sporadic forms of primary hyperparathyroidism (PHPT), and the encoded product parafibromin has been established as a marker for facilitating parathyroid tumour classification. HRPT2 mutations and reduced nuclear expression of parafibromin are readily observed in parathyroid carcinomas but rarely in benign tumours, thereby aiding the identification of malignant PHPT. Recently, parafibromin has been shown to localize to the nucleolar compartment, and nucleolar parafibromin exhibits tumour-suppressive properties in vitro. In this study, nucleolar parafibromin immunoreactivity was assessed by high-power magnification microscopy in 82 parathyroid tumours previously analysed for nuclear parafibromin, including 23 carcinomas, 16 atypical adenomas, and 43 adenomas. Absent nucleolar expression was evident in three carcinomas and in one atypical adenoma, which also showed expression of nuclear parafibromin in all or subsets of the tumour cells. All three carcinomas carried HRPT2-inactivating mutations predicted to abolish the three nucleolar localization signals of parafibromin. The demonstrated absence of nucleolar parafibromin in three carcinomas with HRPT2 mutations suggests that parafibromin exhibits nucleolar tumour suppressor properties also in vivo, and disruption of nucleolar localization might propel parathyroid tumorigenesis independent of nuclear parafibromin expression. The loss of nucleolar staining in the presence of nuclear parafibromin suggests that parafibromin immunoreactivity should also be assessed in the nucleoli, as the sensitivity for the detection of malignant and atypical PHPT is increased compared to scoring of nuclear parafibromin alone.