The involvement of protein kinase C in chemotaxis of normal dermal fibroblasts to a mitogenic and a non-mitogenic attractant was investigated. Neomycin, an inhibitor of phosphoinositide metabolism, H7, staurosporine, and sphingosine, inhibitors of protein kinase C, as well as amiloride, an inhibitor of the Na+/H+-antiport, all abrogated chemotaxis of fibroblasts to platelet-derived growth factor and to a chemotactically active fragment of fibronectin. Down-regulation of protein kinase C by phorbol ester likewise diminished the capacity of fibroblasts to move directionally to these attractants. Therefore, all three of these signal transduction steps are required for the chemotactic response of this type of cells.