The neuronal uptake of norepinephrine (NE) in sympathetically innervated tissues is mediated by a high-affinity NE uptake transporter (NET). Rat interscapular brown adipose tissue (ISBAT) is densely innervated by the sympathetic nervous system for the control of cold- and diet-induced thermogenesis. To determine if cold exposure regulates the NET, kinetic parameters for [³H]NE uptake and [³H]nisoxetine (Nis) binding were determined in ISBAT from 7-day cold-exposed (CE) and control rats. Uptake of [³H]NE in ISBAT slices was of high affinity (1.6 µM). After 7 days of cold exposure the affinity for [³H]NE uptake was not altered; however, the uptake capacity was decreased (38%) in ISBAT slices from CE rats. Kinetic parameters for [³H]Nis binding demonstrated a single high-affinity site in ISBAT from CE and control rats with similar affinity. The density of [³H]Nis sites in ISBAT was decreased (38%) following cold exposure. A time course (2 h-7 days) for cold exposure demonstrated downregulation of [³H]Nis binding density by day 3, which remained through day 7. The affinity for [³H]Nis binding was transiently decreased at 2 h of cold exposure. Similarly, ISBAT NE content was decreased at 2 h of cold exposure. Pair feeding CE rats to food intake of controls normalized plasma NE content; however, [³H]Nis binding density in ISBAT remained decreased in pair-fed rats. These results demonstrate that the ISBAT NET is downregulated following cold exposure. Reductions in ISBAT NE content precede alterations in NET density; however, plasma NE content is not related to regulation of the NET.