Brown Norway (BN) rats exhibit a selective deficit in the hypercapnic ventilatory response (HVR) compared to Dahl Salt‐sensitive (SS) rats, and show differential expression of various pH‐sensitive channels in postulated sites of central CO2/H+chemoreception, including the serotonergic medullary raphé and nucleus of the solitary tract (NTS). Here, we test the hypothesis that brainstem neuromodulators, such as serotonin (5‐HT), dopamine (DA), and norepinephrine (NE), and their metabolites, are altered in BN rats compared to SS rats. To test this hypothesis, we rapidly removed whole brains from adult (>7 weeks of age) BN (n=6) and SS (n=6) rats, and separated it into 5 major brain regions: 1) medulla, 2) pons/midbrain, 3) cerebellum, 4) hypothalamus and 5) forebrain for high‐performance liquid chromatography (HPLC) analysis. In separate animals, we transcardially‐perfused and fixed brains for cryostat sectioning and immunostaining for 5‐HT (TPH‐expressing) or TH‐expressing neurons. We found that tissue levels of 5‐HT were significantly (p<0.05) lower in BN medulla, cerebellum and hypothalamus, and 5‐HIAA levels were lower (p<0.05) in medulla, pons/midbrain, hypothalamus and forebrain compared to SS rat tissues. In addition, tissue NE levels were also lower (p<0.05) in the hypothalamus of BN rats, but not different in any other region compared to SS rats, and we found no differences in DA in all tissues studied. Despite the reduced 5‐HT, 5‐HIAA, and NE levels, we did not find a difference in counts of TPH+ or TH+ neurons throughout the medulla. These data suggest deficiencies in 5‐HT and NE may be related to altered neuronal function rather than altered cell numbers in BN rats, which may contribute to the deficit in the HVR in BN rats. Supported by NIH HL097033 and Veterans Affairs.