Highly invasive cell subpopulations from a human prostate carcinoma cell line, PC-3, were selected for by allowing the parental PC-3 cells to invade through reconstituted basement membrane, Matrigel. These cells were collected, cultured and then selected further by repeated invasion through the in vitro invasion chamber. The invasive subpopulations (I-PC3 (2) and (3)) were found to be approximately 15-fold more invasive in vitro than the parental cells, had a distinct rounded morphology in culture, and proliferated more rapidly than the parental cells. When injected either subcutaneously or intraperitoneally into immunocompromised SCID mice, the I-PC3 cells were found to form tumors at the primary sites and to be highly invasive and metastatic. In contrast, the parental PC-3 cells formed tumors at the site of inoculation in these mice but failed to invade or metastasize. The I-PC3 cells attached equally as well as PC-3 cells to fibronectin, laminin, collagen type IV and vitronectin, but unlike the parental PC-3 cells these invasive variants failed to spread on any of these substrates. On Matrigel, the PC-3 cells became highly organized, whereas the I-PC3 cells remained rounded, clumped together and penetrated into the Matrigel. Biochemical analysis of the expression of adhesion proteins and integrins demonstrated that whereas the parental cells synthesized and secreted substantial amounts of fibronectin, the I-PC3 cell variants did not secrete any fibronectin. Although both PC-3 and I-PC3 cells expressed equivalent levels of cell surface alpha v beta 3, alpha 2 beta 1 and alpha 5 beta 1 integrins, the expression of the alpha 3 beta 1 integrin, which is expressed at very high levels on the parental PC-3 cells, was drastically reduced on the invasive I-PC3 cells. This decrease in expression of alpha 3 occurred also at the level of mRNA expression. Finally, whereas the PC-3 cells express alpha 6 beta 1, in the invasive I-PC3 cells the alpha 6 subunit was associated mostly with the beta 4 subunit. Since the alpha 6 beta 4 integrin is analogous to the A9 tumor antigen which is associated with aggressive human squamous cell carcinomas, the apparent overexpression of alpha 6 beta 4 may also participate in the aggressive behavior of these variant prostate carcinoma cells. Alterations in the expression of the alpha 3 beta 1 and alpha 6 beta 4 integrins may thus allow these cells to become more invasive, and lead to an increased propensity for metastasis.