The sequence of molecular changes leading to neoplastic transformation in the gallbladder remains elusive. The aim of this study is to characterize the spectrum of nuclear p16 protein product immunohistochemical expression in tissue microarray cores taken from resected gallbladders, comprising histologically normal gallbladder epithelia (n = 29), dyplastic epithelia (n = 19), reactive atypia (n = 7), and gallbladder adenocarcinoma (n = 23). Nuclear staining for p16 was evaluated for intensity (range, 0-3) and distribution (range, 0-3), and a summary staining score (range, 0-6) was obtained. Pairwise comparisons were significant for increased p16 protein expression in dysplastic epithelium (6/19, 31.6%) and invasive carcinoma (9/23, 39.1%) when compared to normal epithelium (0/29, 0%) (P = .003 and P = .0006, respectively). A quantitative increase of nuclear expression of p16 in reactive atypia (1/7, 14%) when compared to normal was also statistically significant (P = .049). Our data demonstrate that nuclear p16 expression is absent in normal gallbladder epithelium and is a frequent event in high-grade dysplasia of the gallbladder and gallbladder adenocarcinoma.