The biosynthesis of angiotensin II-like peptide was measured in primary cultured brain cells from the fetal rat. Cells from the whole brains of 20-day gestational age Sprague-Dawley rats were dissociated by mild trypsinization and grown for 5 days in supplemented (serum-free) medium prior to experimental analysis. The time-dependent incorporation of [3H]proline into newly synthesized angiotensin II-like peptide was measured by radioimmunoassay using specific angiotensin II antisera. Analysis of radioimmunoassay data revealed an increase in the amount of tritium-labeled angiotensin II in the crude extract of the brain cells during the first 24 h in culture. The chromatographic character of the angiotensin II-like peptide was further identified by high pressure liquid chromatography. Elution profiles for newly synthesized angiotensin II were identical to those profiles generated for [3H]angiotensin II and nonradiolabeled angiotensin II standards. To determine the bioactivity of the angiotensin II-like peptide, fractions of the column-purified peptide were injected into the region of the rat lateral ventricle via an indwelling cannula. Systolic pressure increased up to 20 mm Hg depending upon the amount of peptide injected. These data clearly support the existence of an endogenous renin-angiotensin system is dissociated brain cell cultures from the fetal rat, and provide an experimental model for further analysis of the regulatory mechanisms of angiotensin II synthesis.