Abstract <p>The endogenous neuromodulator taurine is involved in the centralregulation of numerous physiological processes. However, the functionalsignificance of taurine in the medial prefrontal cortex (mPC), akey region for decision-making and emotion control, remains poorlyunderstood. Using Sprague–Dawley rats, we investigated the roleof taurine signaling in the mPC and the possible contribution oftaurine–serotonergic interactions to fear memory generalization.As a fear model, we used a conditioned fear response (CFR) withdifferentiation of auditory cues—a conditioned cue (CS+) reinforcedby footshocks (CFR Training) and a safe differentiation cue (CS–)(Differentiation). Extracellular serotonin (5HT) and taurine levelsin the mPC were monitored by in vivo microdialysis coupled withhigh‑performance liquid chromatography. We found that CFR Trainingtriggers taurine release in the mPC, which is absent during differentiation. Administrationof 1 mM taurine into the mPC decreased basal 5HT levels in the mPCand reduced 5HT levels during CFR Training and Differentiation 1tests. This treatment did not affect CFR acquisition or its primarygeneralization, as it did not alter the duration of freezing (amarker of pain stimulus expectancy) to CS+ and CS– on the day ofCFR formation. Taurine administration into the mPC decreased CFRgeneralization 24 h later, manifested as reduced freezing durationto CS– in the Differentiation 2 test. Freezing to the unreinforcedCS+ during CFR testing and anxiety‑like behavior in the elevatedplus maze were unchanged after taurine administration. These noveldata indicate, first, a natural enhancement of intercellular taurinesignals in the mPC during CFR Training. Second, they demonstratean inhibitory effect of taurine administered into the mPC on theformation of generalized fear memories and suggest a possible involvementof taurine–serotonergic influences in this process. The resultsexpand our understanding of pharmacological means for modulatinggeneralized fear.</p>