An in vitro system has been established in which conversion from non-metastasizing to metastasizing adenocarcinoma cells can be induced, and subsequently subjected to analysis of the expression of proteases and tissue inhibitor of metalloproteinases-1 (TIMP-1). A human salivary-gland adenocarcinoma cell clone HSGc, with no metastatic ability, was exposed to N-methyl-N-nitrosourea (MNU). Following exposure to MNU, cells with altered morphology were cloned. Upon s.c. inoculation into nude mice, MNU-treated HSGc clones formed metastatic foci in various organs, and then 5 metastasizing clones were isolated. Evaluation of expression of tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), metalloproteinases and TIMP-1 was performed by means of enzyme immunoassay, zymogram, or immunoblot. MNU-treated HSGc and metastasizing clones were found to secrete high levels of tPA, while HSGc produced undetectable levels of this enzyme. Expression of uPA was not observed in any of the cell clones. When the secretion of gelatinolytic enzymes was examined, metastasizing clones produced higher levels of 57- and 32-kDa, but not of 92- or 72-kDa gelatinases, as compared to HSGc cells. Although TIMP-1 was detected in all cell clones, metastasizing clones secreted less TIMP-1 than HSGc cells; in addition, one metastasizing clone produced TIMP-1 with a molecular weight distinct from that of 28-kDa TIMP-1. Our results suggest that the acquisition of metastatic ability by human salivary-gland tumor cells is closely associated with increased secretion of several metalloproteinases as well as decreased or altered TIMP-1 expression.