We have previously shown that protein kinase C (PKC) ζ and/or PKCδ are necessary for endothelin-1 (ET-1)-induced human myometrial contraction at the end of pregnancy (Eude I, Paris P, Cabrol D, Ferré F, and Breuiller-Fouché M. Biol Reprod 63: 1567–1573, 2000). Here, we report that the selective inhibitor of PKCδ isoform, Rottlerin, does not prevent ET-1-induced contractions, whereas LY-294002, a phosphatidylinositol (PI) 3-kinase inhibitor, affects the contractile response. This study characterized the in vitro contractile response of cultured human pregnant myometrial cells to ET-1 known to induce in vitro contractions of intact uterine smooth muscle strips. Cultured myometrial cells incorporated into collagen lattices have the capacity to reduce the size of these lattices, referred to as lattice contraction. Neither the selective conventional PKC isoform inhibitor, Gö-6976, or rottlerin affected myometrial cell-mediated gel contraction by ET-1, whereas this effect was blocked by LY-294002. We found that treatment of myometrial cell lattices with an inhibitory peptide specific for PKCζ or with an antisense against PKCζ resulted in a significant loss of ET-1-induced contraction. Evidence is also presented by using confocal microscopy that ET-1 induced translocation of PKCζ to a structure coincident with the actin-rich microfilaments of the cytoskeleton. We have shown that PKCζ has a role in the actin organization in ET-1-stimulated cells. Accordingly, our results suggest that PKCζ plays a role in myometrial contraction in pregnant women.