We tested two hypotheses about monoamine neurotransmitters in two strains of rats that differ in their sensitivity to obesity when eating a high-fat diet; 1) that the concentrations of norepinephrine and serotonin and of their metabolites differ in the extracellular fluid of the ventromedial hypothalamus of conscious, unrestrained Osborne-Mendel and S 5B/Pl rats, and 2) that these monoamines are altered differently between strains by a high-fat diet. The monoamines were measured by HPLC in dialysate collected by in vivo microdialysis in rats eating a semisynthetic low-fat diet (10% of kcal as fat) and again after either two or seven days of eating a high-fat diet (56% of kcal as fat). Norepinephrine, serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5-HIAA) were lower in Osborne-Mendel rats than in S 5B/Pl rats eating the low-fat diet. Norepinephrine and serotonin both increased in Osborne-Mendel rats with the onset of the high-fat diet so that Osborne-Mendel and S 5B/Pl rats no longer differed in these neurotransmitters. By day 7 of high-fat feeding, the concentrations of 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-HIAA and the 5-HIAA/5-HT ratio rose in both strains. Ambient extracellular monoamines in the medial hypothalamus are lower in Osborne-Mendel rats than in S 5B/Pl rats and the response of these catecholamines to dietary fat was greater in Osborne-Mendel rats than in S 5B/Pl rats.