A direct radio-immunoassay (RIA) for renin substrate (RS) was compared to the enzymatic (indirect) assay, which measures intact RS only, whereas a direct assay measures both intact RS and des-angiotensin I-RS. In normal subjects, a significant, albeit weak, correlation between the methods was noticed. In hypertensive patients with different levels of plasma renin activity (PRA), RS concentration measured by both assays increased with increasing PRA, and for patients with PRA greater than 10 micrograms AI/l/h, the direct assay gave significantly higher RS values (55%), compared to the enzymatic assay. This indicates consumption of RS by increasing plasma renin and increasing production rate of RS with increasing PRA, of potential importance in the pathogenesis of hypertension. In 11 patients with renovascular hypertension, treatment with the angiotensin-converting enzyme (ACE) inhibitor, lisinopril, resulted in a significant increase in PRA, accompanied by a decrease in RS measured by the enzymatic assay. Lowered plasma RS concentration, by reduction of the velocity of the renin-RS reaction, will distort and invalidate results of PRA determination during treatment with ACE inhibitory compounds. No change in RS measured by direct RIA was noticed, however. The results suggest that ACE inhibition may not have an effect upon RS production and that its effect on plasma RS is limited to a reduction of intact RS measured by the enzymatic assay.