Increased protein kinase C (PKC) activity in malignant breast tissue and in most aggressive breast cancer cell lines has suggested a possible role of PKC in breast carcinogenesis and tumor progression. We have investigated here the involvement of PKC in the in vitro invasiveness and motility of several breast cancer cell lines. Modulation of PKC activity by treatment with a phorbol ester (TPA), drastically increased the invasiveness of 2 estrogen receptor-positive (ER+) lines (MCF7 and ZR 75.1), whereas it markedly decreased the invasiveness of 2 ER- cell lines (MDA-MB-231 and MDA-MB-435). A PKC inhibitor (H7) reversed the TPA effects in MCF7 cells, whereas it mimicked TPA action in MDA-MB-231 cells. All of these effects of TPA also were observed to a similar extent for cell chemotaxis, and they were not dependent on protein neo-synthesis. In parallel, short TPA treatment induced cell spreading and microtubule organization in MCF7 cells and inverse morphological changes in MDA-MB-231 cells. In ER+ cells, constitutive PKC activity and PKCalpha expression were very low as compared to ER- cells, and this correlated with the invasive potential of the cells. The opposed effects of TPA in ER+ and ER- cells could be due to the abnormal TPA regulation of PKCalpha observed in ER- cells.