We previously reported NF2 mutations in malignant mesothelioma (MM) cell lines and corresponding primary tumors. We have now generated polyclonal antibodies that specifically recognize the C-terminus of the NF2 protein. Western blot analysis was performed on 25 MM cell lines, 14 of which showed no NF2 expression. Single-strand conformation polymorphism and DNA sequence analyses revealed NF2 mutations in each of these 14 cell lines. To explore the mechanism of inactivation of NF2, loss of heterozygosity analysis was performed with two microsatellite markers located in the vicinity of the NF2 locus in chromosome band 22q12. Eighteen of the 25 cell lines (72%) showed losses at one or both loci tested. All cases exhibiting mutation and/or aberrant expression of NF2 showed allelic losses, suggesting that inactivation of NF2 in MM occurs via a two-hit mechanism.