LIMD1 is a member of the ZYXIN family of related proteins which includes AJUBA, TRIP6, LPP, WTIP, migfilin, and ZYXIN. The LIMD1 locus, 3p21.3, has been shown to undergo loss of heterozygosity in neoplastic tissues, suggesting potential tumor suppressor function. To further understand the role of LIMD1 in cancer, we have characterized endogenous expression of the LIMD1 protein and evaluated LIMD1 RNA expression in primary human breast tumors. LIMD1 levels were found to be constant throughout the cell cycle, but LIMD1 is phosphorylated during mitosis in HeLa cells. In addition, we observed colocalization of endogenous LIMD1 with vinculin at focal adhesions. In the MDA-MB435 cell line, which lacks LIMD1 expression, we detected methylation of the putative promoter region. LIMD1 mRNA expression was found to vary among primary human breast tumors; however, differences in LIMD1 expression in human breast cancers were not associated with DNA methylation of the predicted promoter region. These results suggest that some breast tumors have altered expression of LIMD1 RNA and that LIMD1 may be involved in cell anchoring via focal adhesions and in the cell cycle, particularly during mitosis.