In order to investigate the endothelin (ET) levels and their relationship to various hormones during the menstrual cycle, we measured endothelin-1, -2 and -3 (ET-1, -2 and -3), big ET-1 and big ET-3 levels in 27 normally cycling women (mean age 27 years). Simultaneous determination of luteinizing hormone-releasing hormone (LH-RH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, progesterone, 17alpha-hydroxyprogesterone (17-OHP), estrone, 17beta-estradiol, delta4-androstenedione, testosterone, active renin, angiotensin-II (A-II) and atrial natriuretic peptide (ANP) in blood, and LH, estrone and 17beta-estradiol and pregnanediol levels in urine was made in the same 27 women. The levels of ET-2 in plasma were found to be 20% of those of ET-1. In addition, ET-1 levels were measured in the endometrium of the normal uterus. Plasma ET-1 and ET-3 levels fluctuated during the menstrual cycle, with a peak at the luteal phase, but showed only a negative relationship (p < 0.01) to each other at the menstrual phase, whereas big ET-1 and big ET-3 levels showed no significant changes. Plasma ET-1 and ET-3 levels showed no significant relationship to the big ETs. As for the relationship to other hormones, plasma ET-3 had a negative relationship (p < 0.01) to prolactin and a positive correlation (p < 0.01) with ANP during the entire menstrual cycle. Plasma ET-1 and ET-3 showed a partial positive or negative correlation to LH, FSH, prolactin and ANP levels, depending upon the cycle phase, whereas plasma ETs and big ETs were unrelated to other hormones in the blood, and LH, estrone, 17beta-estradiol and pregnanediol in the urine throughout the menstrual cycle. At each menstrual stage, plasma ET-3 levels were more significantly related to LH, FSH, prolactin and ANP than ET-1, indicating a closer relationship between ET-3 and these circulating hormones during the menstrual cycle. The ET-1 level showed no significant change in the endometrium during the menstrual cycle.