Spreading of HeLa cells on a gelatin substratum is initiated by the activation of protein kinase C epsilon (PKC epsilon) upon contact of the cells with the matrix. In this study, we examined the functional role of PKC epsilon in the activation of mitogen-activated protein kinase (MAP kinase) and its relationship to cell spreading. MAP kinase isoforms, Erk-1 and -2, are activated upon attachment of HeLa cells to gelatin. Inhibition of PKC with calphostin C blocked cell spreading without any effect on MAP kinase activation. In contrast, inhibition of MAP kinase kinase blocked adhesion-induced MAP kinase activation, but showed no effect on either translocation of PKC epsilon or cell spreading. Thus, activation of PKC epsilon that occurs upon HeLa cell attachment to gelatin is related to cell spreading but not to the activation of MAP kinase, and MAP kinase is activated upon HeLa cell attachment in the absence of cell spreading.