OBJECTIVE: To determine the direct effects of cigarette smoke (CS) on human endometrial surface epithelial cell line proliferation. DESIGN: In vitro study using HES cells and primary human endometrial cells. SETTING: University-based academic center. PATIENT(S): Premenopausal women in the proliferative phase of the cycle. INTERVENTION(S): The HES cells and primary human endometrial cells were exposed to cigarette smoke-saturated solution. MAIN OUTCOME MEASURE(S): Cell proliferation and expression of different isoforms of nitric oxide synthase. RESULT(S): Cigarette smoke inhibited HES cell proliferation in a dose- and time-dependant manner. The inhibitory effect of CS was blocked by hemoglobin and enhanced by L-arginine (L-Arg). Cigarette smoking and nicotine stimulated the expression of endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) whereas benzo[a]pyrene (BP) only stimulated the expression of eNOS in HES cells. Cigarette smoke stimulated the expression of eNOS/iNOS in primary human endometrial cells, comprised of epithelial and stromal cells. The effect of CS on eNOS/iNOS expression in HES cells was blocked by ascorbic acid but not by glutathione. CONCLUSION(S): Cigarette smoke inhibits endometrial cell proliferation through a nitric oxide-mediated pathway.