Among the unsettled questions in the physiology of human epidermal growth factor (EGF) are (1) does EGF circulate in the blood and (2) what is the source of the abundant urinary immunoreactive EGF (irEGF). Therefore, we monitored the concentration of irEGF by an ultrasensitive assay in blood plasma from 5 healthy subjects every 20 min overnight carefully avoiding activation of platelets. Detectable levels (0.8-3.7 pM) were observed in only one of the subjects, in 5 of 29 samples. In random day-time plasma samples from 18 healthy adults, EGF was undetectable (less than 0.8 pM) in 13 subjects, and in 5 subjects EGF levels ranged from 2.2 to 4.9 pM. Furthermore, in 5 patients with a tumor in a functioning kidney we measured urinary relative irEGF concentration (nmol/mmol creatinine) before and after unilateral nephrectomy. The concentration fell by approximately 50%. Our findings are consistent with (1) blood irEGF residing exclusively in platelets, and (2) urinary irEGF originating from the kidneys.