Recent studies in our laboratory demonstrated that an aqueous extract of Valeriana officinalis rhizomes and roots induces Na+-dependent, Ca2+-independent [³H]GABA release from rat brain synaptosomes via the GABA carrier, with the extract and 2,4-diaminobutyric acid (DABA) releasing [³H]GABA from the cytosolic compartment. To identify the component(s) responsible for this release and explore the involvement of an exchange mechanism, we analyzed the amino acid composition of the extract using HPLC and investigated [³H]GABA release from synaptosomes. The extract was standardized by valerenic acid, and synaptosomes were isolated via centrifugation, incubated with [³H]GABA, and superfused to measure release. HPLC analysis revealed high concentrations of arginine, glutamine, alanine, and GABA (≈5 mM) in the extract. The GABA concentration was sufficient to induce [³H]GABA release via a homoexchange mechanism, as unlabelled GABA (25 μM) elicited a similar release (11.8 ± 2.4% of total vs. 14.3 ± 1.1% for the extract) and their effects were not additive, indicating the same mechanism. High glutamine (≈14 mM) may explain the extract’s sedative effects in vivo, as glutamine crosses the blood-brain barrier and is metabolized to GABA in GABAergic neurons, stimulating GABA synthesis. While GABA’s poor blood-brain barrier penetration argues against central GABAergic modulation, it may mediate peripheral relaxant activity. These findings suggest GABA in the extract is the main driver of [³H]GABA release, with glutamine potentially contributing to sedation.