Homeobox genes regulate sets of genes that determine cellular fates in embryonic morphogenesis and maintenance of adult tissue architecture by regulating cellular motility and cell-cell interactions. Our previous studies showed that a specific member, HOXD3, when overexpressed, enhanced cell motility and invasiveness of human lung cancer A549 cells (Hamada et al. Int. J. Cancer 2001; 93: 516-25 [19]). In the present study, we investigated the roles of HOXD3 in motile and invasive behavior of human malignant melanoma cells. Of seven melanoma cell lines examined here, six cell lines expressed the HOXD3 gene, whereas normal melanocytes did not. We transduced the HOXD3-antisense gene expression vector into two cell lines (A375M and MMIV). The cell transduced with the HOXD3-antisense gene showed reduced in vitro invasion of Matrigel. The transduction of the HOXD3-antisense gene also decreased cell spreading, haptotactic activity to vitronectin and laminin-1, and phagokinetic activity. To find the difference of gene expression between the HOXD3-antisense-transduced A375M cells and the control A375MNeo2 cells, we carried out cDNA microarray analysis. The results of the microarray analysis indicated that the increased expression of cdc42-interacting protein 4, KIAA0554 and tropomyosin 1, which are all associated with the cytoskeletal system, may be involved in the reduction of motile and invasive activity by the HOXD3-antisense gene transduction.