The neural substrates of fear conditioning in rats have been well characterized, with converging lines of evidence indicating that conditioned stimulus (CS) and unconditioned stimulus (US) information form a CS–US association in the amygdala. Auditory CS information can reach the amygdala via two routes: a direct thalamo-amygdala pathway, and an indirect thalamo-cortico-amygdala pathway. Although either pathway can fully support learning when the alternate pathway is disrupted, many studies to date have argued that the thalamo-amygdala pathway is the principal auditory CS pathway in intact brains. To test this hypothesis, we trained rats in auditory fear conditioning, and 24 h later lesioned either pathway, leaving the alternate pathway intact. Later, animals were tested for conditioned freezing to the auditory CS. We report that lesions of the thalamo-amygdala pathway produced severe but incomplete deficits in freezing during the tone retention test, while lesions of the thalamo-cortico-amygdala pathway completely abolished freezing during tone presentation. These results suggest that the thalamo-cortico-amygdala pathway is the principal auditory CS pathway when the brain is intact.