Pituitary tumors develop at a high frequency in retinoblastoma (Rb)-knockout mice; however, defects in the Rb gene are not common in human pituitary tumors. The inverse correlation of Rb and p16 defects in certain human tumors has led us to investigate the expression of p16 in human pituitary tumors as an indirect mechanism of Rb inactivation. By Western blot analysis, the p16 gene product was undetectable in 25 human pituitary tumors, whereas high levels of p16 could be demonstrated in 10 normal human pituitary specimens under the same conditions of protein extraction and immunoblotting. Similar results were obtained at the mRNA level with low to undetectable levels of p16 mRNA in 13 of 14 pituitary tumors relative to 5 normal pituitary specimens. Single-strand conformation polymorphism analysis of p16 exons 1 and 2 revealed no mobility shifts in 25 tumors; however, a quantitative differential PCR analysis revealed diminished amplification of p16 relative to a control gene in 3 of 25 tumors, suggesting homozygous p16 gene loss. We conclude that altered expression of the p16 gene product occurs at a high frequency in human pituitary tumors. This altered expression is not associated with frequent p16 mutation or gene loss, suggesting that alternative mechanisms of gene inactivation and/or altered regulation occur in the majority of these tumors.