Metastasis is a complicated multistep process that involves interactions between cancer cells and their surrounding microenvironments. Previously, we have established a series of lung adenocarcinoma cell lines with varying degrees of invasiveness. Tracheal graft assay confirmed that cell lines with higher in vitro invasiveness had greater in vivo invasive potential. In this study, we used these model cell lines to identify invasion-associated genes using cDNA microarray with colorimetric detection. A more invasive subline, CL 1-5-F 4, derived from metastatic lung tumor of severe combined immunodeficient mice inoculated with CL 1-5 cells, was combined with CL 1-0, CL 1-1, and CL 1-5 in cDNA microarray screening. cDNA microarray membranes, each containing 9600 nonredundant expressed sequence tag clones, were used to identify differentially expressed genes in these cell lines. For statistical analysis, self-organizing map algorithm was performed to identify the expression patterns. Positive correlation between gene expression levels and cell line invasiveness was found in 2.9% of the 9600 putative genes. On the other hand, negative correlation was found in 3.3% of the genes. The trends of expression of some of the genes were also confirmed by Northern hybridization and flow cytometry. Our data demonstrated that genes related to cell adhesion, motility, angiogenesis, signal transduction, and some other expressed sequence tag genes may play significant roles in the metastasis process. These results substantiate the model system with which one can identify invasion-associated genes by using cDNA microarray and cancer cell lines of different invasiveness. This technique may allow us to explore complex interactions between multiple genes that orchestrate the process of cancer metastasis.