Overexpression of membrane-type matrix metalloproteinase (MT-MMP-1) results in the activation of both endogenous and exogenous 72-kDa gelatinase. To understand the effects of MT-MMP-1 on 72-kDa gelatinase activation, we analyzed its expression in human fibroblasts and HT-1080 fibrosarcoma cells. Both cell types expressed the MT-MMP-1 mRNA constitutively at a considerable level and treatment of cells with PMA enhanced the expression about 2-3-fold. Concanavalin A treatment increased MT-MMP-1 mRNA levels in fibroblasts about 4-fold. Induction of MT-MMP-1 by phorbol 12-myristate 13-acetate (PMA) required protein synthesis as shown by cycloheximide inhibition. The induction was also inhibited by dexamethasone. Analysis of MT-MMP-1 mRNA stability using actinomycin D indicated that the half-life was rather long and not affected by PMA, suggesting transcriptional regulation. Only HT-1080 cells had significant 72-kDa gelatinase processing activity after treatment with PMA or concanavalin A, while fibroblasts were virtually negative. Immunoblotting analysis of fibroblast lysates indicated that MT-MMP-1 was present mainly in a 60-kDa form. PMA and concanavalin A caused 2-4-fold increases in its protein levels, while in HT-1080 cells PMA, concanavalin A, or overexpression of MT-MMP-1 did not significantly enhance the level of the 60-kDa protein. Instead, an immunoreactive, proteolytically processed 43-kDa form was observed, and its appearance correlated to 72-kDa gelatinase processing activity. Thus 72-kDa gelatinase activation, while enhanced by MT-MMP-1 expression, needs additional co-operating factors.