New assay methods have been devised to quantitate tumor cell invasion of tissues of differing histological complexity maintained as organ cultures in vitro (chorioallantoic membrane of chicken, mouse urinary bladder, and canine blood vessel). In addition to quantitating tumor cell invasion, these methods also allow recovery of invasive cells for comparison with noninvasive cells. These methods have been used to select variant sublines from murine B16-F1 and B16-F10 melanoma lines that display significantly greater tissue-invasive abilities than the parent lines. B16 variant sublines selected in vitro for increased invasiveness through the bladder wall or vein also show a significant increase in their ability to form spontaneous and experimental metastases in vivo. In contrast, cells from the same parent cell line selected for increased invasiveness through the chorioallantoic membrane do not show significant alterations in metastatic behavior. We conclude that invasive variants can be isolated from the parent B16 tumor by several in vitro methods and that the level of expression of the invasive phenotype in vivo may be determined by the severity of the selection procedure in vitro.