We have recently reported the presence of inducible nitric oxide synthase (iNOS) in the human breast cancer cell line ZR-75-1. The purpose of the present study was to examine differences in expression of endothelial (eNOS) and inducible nitric oxide synthase in normal human mammary epithelial cells (HMEC) compared with two variants of the ZR-75-1 cell line. One variant has acquired estrogen independence, the other has acquired resistance to tamoxifen. Immunohistochemical investigations demonstrated that 100% of HMEC cells staining positive for both eNOS and iNOS. ZR-75-1 cells showed 100% staining for eNOS and 52% positive staining for iNOS. There was no difference in staining between the parent cell line and cells which had acquired resistance to tamoxifen (ZR-75-9a1). However, in the breast cancer cell line which had acquired estrogen independence (ZR-PR-LT), less than 5% of cells exhibited positive staining for eNOS and staining for iNOS was undetectable. L-Arginine increased NO production in both ZR-75-9a1 and ZR-PR-LT cells. Progesterone was able to down regulate NO production in both ZR-75-1 and ZR-75-9a1 cells and this effect was reversible by RU486. These results support the suggestion that loss of NOS expression may be associated with the progression of breast cancers.