As a model for hormone-dependent breast cancer, we studied the MCF-7 cell line to examine differences in the stimulation of the inducible (i) and endothelial (e) nitric oxide synthase (NOS) and the role of 17beta-estradiol (E(2)). MCF-7 cells were stimulated with (a) E(2) (10(-8)M) and (b) a combination of different cytokines such as interleukin-1 beta (Il-1beta), tumor necrosis factor alpha (TNF-alpha) and interferon gamma (INF-gamma), and lipopolysaccharide (LPS). e-NOS and i-NOS proteins were measured using Western blot analysis. Using the Griess method nitric oxide (NO) was estimated by assessing the stable product nitrite (NO(2-)) in the culture medium, and a direct method, employing EPR spin trapping also was used. Western blot analysis revealed the presence of e-NOS and i-NOS in MCF-7 cells. In Western blot analysis, e-NOS, but not i-NOS, expression could be stimulated by E(2). An increase in NO(2-) was noted after stimulation of MCF-7 using different combinations of cytokines Il-1beta, TNFalpha and INFgamma, and LPS, but not after E(2). In conclusion, e-NOS and i-NOS are weakly expressed in the MCF-7 cell line, but are stimulated differently. The MCF-7 cell may contain both a constitutive NOS and an inducible NOS.