We have previously shown that during wound healing migrating keratinocytes, which are in contact with the dermal matrix, express interstitial collagenase, whereas basal epidermal cells, which reside on an intact basement membrane, do not. Duplicating this in vivo pattern, collagenase production was induced in primary human keratinocytes grown on native type I collagen, but only background levels of enzyme were detected in cells cultured on denatured type I collagen or on Matrigel. Using genistein, herbimycin A, and sodium orthovanadate, we show that tyrosine kinase activity was required for collagen-mediated induction of keratinocyte collagenase. Similarly, collagenase steady-state mRNA levels and the activity of a transfected human collagenase-promoter CAT construct were inhibited by genistein and enhanced by orthovanadate. Staurosporine and H-7 also blocked collagenase production, indicating that protein kinase C activity was also required for collagen-mediated induction of keratinocyte collagenase. All inhibitory effects were dose-dependent, and no compound significantly affected total protein synthesis. Furthermore, both tyrosine kinase and protein kinase C inhibitors blocked phorbol ester-mediated induction of collagenase, but only protein kinase C antagonists abrogated phorbol ester-mediated induction of c-fos mRNA. These data suggest that similar signal transduction pathways are used by various agonists to mediate the stimulation of interstitial collagenase production.