In the multistep process of tumor metastasis, different cellular functions are known to be influenced by the urokinase plasminogen activator (u-PA). In different types of malignancies, u-PA has been shown to correlate strongly with a malignant tumor cell phenotype. Besides its proteolytic activity, the enzyme is effective by signal transduction mechanisms. To elucidate u-PA functions in osteosarcoma, in the present study, the osteosarcoma cell line MNNG/HOS was transfected with an antisense (as) expression vector encoding the 3' end of u-PA-cDNA. Several stably transfected cell clones were characterized and compared with the parental cell line. The antisense transfection resulted in: (1) stable incorporation of the vector construct into cellular genome, as demonstrated in Southern blot; (2) decreased u-PA expression in Northern blot; (3) 50% reduced u-PA protein expression in both cell homogenate and cell culture medium; (4) unchanged cellular proliferation and u-PAR (urokinase plasminogen activator receptor) expression. In comparing functional analysis, as-clones showed (I) significant reduced in vitro invasion and motility (chemotaxis with collagen I); (II) significantly reduced adhesion activity to both vitronectin and collagen I matrices but unchanged adhesion on matrigel; (III) reduced in vivo metastasis in chick embryos after i.v.-application. All together, this data show that malignancy of the osteosarcoma cell line MNNG/HOS is positively influenced by urokinase in terms of migration and selective adhesion. Both effects were observed besides the previously described enzyme functions in tumor cell invasion and basement membrane degradation.