Summary. Sheep plasTiia renin substralf was purified 1,200-fold l>y usinjj nephrfctomised sheep plasma, followed by DEAE-Sephadex chromatojijraphy and gel filtration. Th? purified substrate contained 8 nji angiotensin Il/nig protein and had an estimated molecular vveijjht of 52,000. The kinetic characteristics of the purified substrate were identical both to those of tuipurified nephrectomised sheep plasma and to normal sheep plasma substrates. At pH 7-5, K,,, of the human renin-sheep substrate reaction was 0-29 [iM and for sheep reninsheep substrate. 2-0 fiM. Sheep substrate was .susceptible to peptic diHeslion with generation of pepsitenshi.Hunuin renin substrate was less readily purified. DEAE-Sephadex chromatography of plasma from pregnant women at 36-40 weeks' gestation produced a 70-fold increase in purity (0-9 ng angiotensin Il/mg protein). No further increase was achieved with gel filtration. Human renin substrate behaved as a larger (mol. wt. 82.000) more anionic protein than sheep substrate and was resistant to the proteolytit actions of both pepsin and shi-ep renin. K,,, for the human renin-human substrate reaction was high and conid not be accurately detennined (range 3-8 nM. mean 5-7 ^iM). The presence of human substrate in a human rcnin-sheep substrate system did not alter the measured initial velocity.In both shet'p and man, the nonnal concentration (»f ri^nin substrate is considerably less than K,,, and must therefore be considered a dtterminant of angiotensin production rate j>i vivo.