Targeted disruptions of the retinoblastoma (Rb) gene result in a high frequency of pituitary tumors in heterozygous mice. While our group and others have reported that loss of heterozygosity (LOH) at the Rb locus in human pituitary tumors is rare, these studies have not excluded small inactivating Rb‐gene abnormalities more frequently found in human tumors and undetectable by LOH‐PCR assays. As a more sensitive means of detecting evidence of these lesions, we have performed Western‐blot analysis of several human pituitary tumors to identify Rb loss at the protein level as well as truncated forms of the Rb protein frequently associated with Rb‐gene mutations. In 24 tumors, Rb protein was detected at levels 1.4‐ to 3.9‐fold those detected in normal post‐mortem pituitary. There was no evidence of truncated forms of the Rb protein and only the hypophosphorylated (active) form of the protein was detected in normal and in pituitary tumor specimens. To investigate the possibility of loss of function mutations in certain tumors resulting in the expression of stable, mutant, hypophosphorylated Rb protein, we further performed SSCP analysis of exons 20 through 24 corresponding to the pocket domain of the Rb protein. Of 20 pituitary tumors examined, no mobility shifts could be demonstrated in this analysis. Our findings provide further evidence that primary Rb inactivation is not common in human pituitary tumors. Our detection of only the hypophosphorylated form of the Rb protein probably reflects the low proliferative state of these tumors. © 1996 Wiley‐Liss, Inc.