When outbred Sprague--Dawley rats are placed on a diet relatively high in fat and calories (HE diet), half develop diet-induced obesity (DIO), while the rest are diet-resistant (DR). When fed a low fat chow diet from weaning, DIO- and DR-prone rats weigh the same, but DIO-prone rats have a number of abnormalities of neural function, many of which are normalized when they become obese after chronic exposure to a HE diet. Because of its important role in the regulation of energy homeostasis in the hypothalamus, we examined the ratio of serotonin (5-HT) to its metabolite, 5-hydroxyindolacetic acid (5HIAA), as an index of transmitter turnover in micropunches from various brain areas in these rats. While still on chow, both DIO- and DR-prone rats showed lower 5-HT turnover in most brain areas sampled during the last hour of the light phase, when animals become active and begin foraging for food, as compared to the first hour of the light phase, when animals are generally quiescent and not eating. However, unlike DR-prone rats, DIO-prone rats did not show a significant time-dependent difference in 5-HT turnover in either the arcuate or paraventricular hypothalamic nuclei. When fasted for 48 h, both DIO- and DR-prone rats showed a generalized 16--46% decrease in 5-HT turnover in the dorsomedial nucleus, perifornical lateral hypothalamus, dentate gyrus and motor cortex as compared to their free-fed counterparts. However, fasted DIO-prone rats showed a 53% greater reduction in the ventromedial nucleus turnover than fasted DR-prone rats. Finally, when DIO rats became obese after 14 weeks on the HE diet, their abnormalities in hypothalamic 5-HT turnover at the end of the light phase were normalized. Thus, DIO-prone rats show abnormalities of diurnal and fasting-induced alterations in brain 5-HT turnover which may predispose them to become obese when dietary fat and caloric density are increased. Once obesity develops, these abnormalities, like those of several other hypothalamic transmitters and peptides, are normalized. This may contribute to the persistence of obesity once it develops.