The Boyden chamber assay is widely used for in vitro measurement of the invasive capacity of cells. However, results can be affected significantly if certain precautions are not taken. Using the Boyden chamber assay we investigated in vitro the invasive potential of a variety of human gynecological tumor cell lines to degrade and migrate through the artificial basement membrane matrix Matrigel. However, in the absence of this Matrigel layer large differences were observed in the ability of cells to adhere to, migrate through and attach to the lower side of the filter membranes. These differences were influenced by cell density, degree of directional locomotion, and the size of the filter pores. To adjust for these influences (which are not directly correlated to the capacity of cells to traverse the Matrigel layer), invasion results were corrected for the ability of cells to migrate through the filter membrane. In addition, the invasion of MDA-MB-231 cells was used as an internal standard to compensate for variations in the Matrigel layer between different experiments. Overall, in our experimental set up, the five human breast cancer cell lines were the most invasive (mean invasion +/- SEM relative to MDA-MB-231 invasion: 104.7 +/- 6.1%), the five human ovarian cancer cell lines the least invasive (60.2 +/- 2.2%) and the six human endometrial cancer cell lines showed an intermediate capacity (79.1 +/- 3.5%). In conclusion, the Boyden chamber assay can be used reliably for studying the invasive potential of cells in vitro, if the ability of the cells to migrate through the filter is taken into account, and a reference cell line is included to enable comparison of the data obtained from independently performed experiments on different cell lines.