Gemcitabine, which induces S-phase arrest, and Vinorelbine, which arrests microtubule organization, are two agents that have demonstrate preferred anti-tumor activity. Nitric oxide acts in diverse functions including anti-tumor and anti-pathogenic activities. In this study, we aimed to examine the distribution of immunoreactivities of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in cells of the MCF-7 breast cancer cell line in response to treatment with Gemcitabine (G), Vinorelbine (V) and combination of Gemcitabine and Vinorelbine (G+V). The distributions of iNOS and eNOS were determined by using indirect immunoperoxidase or immunofluorescence methods and ELISA. Cells incubated with G, V and G+V for 24, 48 and 72 h were immunolabelled with anti-eNOS and anti-iNOS primary antibodies. Apoptosis was determined by TUNEL assay. A significant increase of eNOS immunolabelling on MCF-7 cells treated with G and G+V was observed. Apoptotic cells were also detected in G, V and G+V treated MCF-7 cells. The immunolabelling of iNOS was detected in all groups but this immunoreactivity was not different among the groups. In conclusion, while G treatment, induced S-phase arrest, triggered the NOS pathway after treatment of MCF-7 cells, V treatment, arrested microtubule organization and did not change the NOS pathway. Detection of increased eNOS immunolabelling and apoptosis after G treatment of MCF-7 cells could be important to the treatment of human breast cancer. (C) 2009 Elsevier GmbH. All rights reserved.