Histological studies have previously demonstrated an association between mast-cell activation/degranulation and areas of connective-tissue lysis in vivo; in addition, mast-cell extracts have been shown to activate latent forms of collagenase and stromelysin. In the present study we have examined the potential roles of rat mast-cell proteinase (RMCP) I and RMCP II as activators of the precursors of matrix metalloproteinase (MMP)-1 (interstitial collagenase), MMP-2 (gelatinase A) and MMP-3 (stromelysin 1). Both RMCPs I and II activated proMMP-3 by converting the 57 kDa precursor into a 45 kDa polypeptide. The N-terminal amino acid of 45 kDa MMP-3 activated by RMCP II was identified as Phe83. By contrast, only RMCP II activated the 52 kDa proMMP-1 by converting it into a 41 kDa protein and generating the new N-termini, namely Gln80 and Val82. The collagenolytic activity which resulted from this cleavage was only 35% of the full activity, but this could not be augmented by subsequent treatment with MMP-3, the latter being a crucial enzyme for the generation of the fully active MMP-1 with Phe81 at the N-terminus, in conjunction with other serine proteinases. Thus RMCP II activates proMMP-1 via a mechanism different from that reported for the stepwise processing by combinations of other trypsin-like enzymes and MMP-3. ProMMP-2 (pro-gelatinase A) was not activated by either RMCP I or RMCP II, despite processing to smaller products.