Retinoids are vitamin A derivatives, which cause growth inhibition, differentiation and/or apoptosis in various cell types, including some breast cancer cells. In general, estrogen receptor (ER)-positive cells are retinoic acid (RA) sensitive, whereas ER-negative cells are resistant. In this report, we show that ER-negative MDA-MB-231 cells are strongly growth inhibited by retinoids in combination with a PKC inhibitor. While neither RA nor GF109203X (GF) has a significant growth inhibitory effect in these cells, RA+ GF potently suppress proliferation. We found that RA+ GF induce apoptosis, as shown by an increase in fragmented DNA, Annexin-V-positive cells and caspase-3 activation. Apoptosis was also induced by GF in combination with two synthetic retinoids. Expression of phosphorylated as well as total PKC was decreased by GF and this was potentiated by RA. In addition, treatment with GF caused a strong and sustained activation of ERK1/2 and p38-MAPK, as well as a weaker activation of JNK. Importantly, inhibition of ERK but not p38 or JNK suppressed apoptosis induced by RA+ GF, indicating that activation of ERK is specifically required. In support of this novel finding, the ability of other PKC inhibitors to cause apoptosis in combination with RA correlates with ability to cause sustained activation of ERK.