The measurement of renin activity, concentration, and substrate in rat plasma has been performed by radioimmunoassay of the angiotensin I generated during incubation of plasma in vitro. The sensitivity of the method permits measurements of all three renin parameters in small plasma samples, allowing sequential determinations to be performed in individual animals. The specificity of the method has been demonstrated by the complete absence of renin in plasma from nephrectomized male rats, by the identical assay slopes of generated angiotensin and angiotensin I standards, and by the parallel changes in plasma renin activity (PRA) and renin concentration (PRC) measured by bioassay and immunoassay after furosemide administration and water deprivation. Also, radioimmunoassay determination of the pH optimum for renin activity, and of the correlations between PRA and PRC, were identical with those obtained by bioassay. The absolute values for renin substrate were similar to those measured by bioassay. However, the values for PRA and PRC measured by radioimmunoassay were higher than those previously observed by bioassay, due to the greater generation of angiotensin I under the incubation conditions employed in the present method. Serial measurement of all three major renin parameters in plasma permits evaluation of the relative contributions of changes in renin secretion and renin substrate to alterations in the activity of the reninangiotensin system during studies in small animals such as the rat. (Endocrinology90: 422, 1972)