Guaiacol peroxidase (G-Px) was measured in extracts from five sections along the length of human uterus on different days of the menstrual cycle or after menopause. The lower uterine-endocervical region had a significantly higher G-Px content (expressed as enzyme units per g wet tissue) than the other sections, although in postmenopausal patients the G-Px activity was uniformly low in all sections of the uterine cavity. We observed no significant changes in G-Px levels during the menstrual cycle, except, possibly, a decrease around ovulation, which precluded a positive correlation between plasma estrogen levels and uterine G-Px content; such estrogen dependence of G-Px has been previously shown in the rat. In vitro, G-Px was inhibited by estriol and 17 beta-estradiol, marginally inhibited by estrone, and most notably inhibited by the catecholestrogens tested (2-hydroxy-17 beta-estradiol, 2-hydroxy-estriol, and 2-hydroxy-estrone), which were equipotent inhibitors; LH and FSH, progesterone, or cortisol had no effect on G-Px activity. We hypothesize that catecholestrogens are natural substrates and regulations of G-Px activity in the human uterus.