The aim of the study was to investigate the effects of estrogen replacement therapy (ERT) on nitric oxide (NO) activity in healthy postmenopausal women. The study group consisted of 22 postmenopausal women (last menses at least 12 months prior to study entry) who were randomized to receive treatment for 2 months with patches that delivered either 50 micrograms/day of 17 beta-estradiol or placebo in a cross-over design. Blood samples for measurements of serum citrulline and arginine were collected at the start of the study and at the end of each treatment course. Serum citrulline and arginine were measured using high-performance liquid chromatography with fluorometric detection. Arginine levels were significantly lower in the ERT group compared to the placebo group, while citrulline levels did not change. The percentage citrulline/arginine ratio was significantly higher in the ERT group (42.9 +/- 21.6) compared to the placebo group (33.9 +/- 18.5) (p < 0.01). The citrulline/arginine ratio, both at baseline and during either ERT or placebo administration demonstrated a positive linear correlation with body mass index (BMI). No correlations were found between follicle stimulating hormone, estradiol and insulin levels and BMI. No correlations were found between age, time since menopause and baseline arginine and citrulline levels or the citrulline/arginine ratio. These data indirectly demonstrate that transdermal estradiol replacement in postmenopausal women is able to stimulate NO production through the involvement of endogenous L-arginine. A positive linear correlation was found between BMI and the citrulline/arginine ratio, suggesting an additional protective cardiovascular effect in overweight women.