AbstractDAL‐1/4.1B (EPB41L3)is a member of the protein 4.1 superfamily, which encompasses structural proteins that play important roles in membrane processes via interactions with actin, spectrin, and the cytoplasmic domains of integral membrane proteins. DAL‐1/4.1B localizes within chromosomal region 18p11.3, which is affected by loss of heterozygosity (LOH) in various adult tumors. Reintroduction of this protein into DAL‐1/4.1B‐null lung and breast tumor cell lines significantly reduced the number of cells, providing functional evidence that this protein possesses a growth suppressor function not confined to a single cell type. For characterization of the mutational mechanisms responsible for loss of DAL‐1/4.1B function in tumors, the exon–intron structure of DAL‐1/4.1B was examined for mutations in 15 normal/tumor pairs of non–small cell lung carcinoma by single‐strand conformation polymorphism analysis. These studies revealed that small intragenic mutations are uncommon in DAL‐1/4.1B. Furthermore, LOH analysis on 129 informative early‐stage breast tumors utilizing a new intragenic C/T single‐nucleotide polymorphism in exon 14 revealed that LOH resulted in preferential retention of the C‐containing allele, suggesting that allele‐specific loss is occurring. These studies indicate that mechanisms such as imprinting or monoallelic expression in combination with loss of heterozygosity may be responsible for loss of the DAL‐1/4.1B protein in early breast disease. © 2004 Wiley‐Liss, Inc.