Alteration of serotonin (5-HT) levels influences developing thalamocortical afferents (TCAs) in primary somatosensory cortex (SI) of rats and mice. The 5-HT(1B) receptor, present on TCAs during the first postnatal week, may be involved in these effects. The present study asked whether administration of 5-nonyloxytriptamine (NNT), a selective 5-HT(1B) receptor agonist, affects TCA organization in rat SI. Littermates were injected five times daily (5x/day), with either 0.1 mg/kg NNT or vehicle from birth to postnatal day 6 (P-6). Animals were killed on P-6, and their brains were processed for high-performance liquid chromatography (HPLC), cytochrome oxidase (CO) histochemistry, cresyl violet, or demonstration of TCAs by placement of 1,1'-dioctadecyl-3,3,3'' 3'-tetra-methylindocarbocyanine perchlorate (Di-I) on thalamocortical radiations. At P-6, NNT treatment decreased 5-HT levels slightly compared with controls, although this difference was not statistically significant. In NNT-treated rats, the Di-I-labeled vibrissae-related pattern showed a range of effects, from fusion of patches related to mystacial vibrissae in treated animals to a less distinct vibrissae-related pattern in SI barrelfield compared with controls. Staining for CO and Nissl stain in layer IV of SI showed a similar range of abnormalities. These results indicate that the agonist action of NNT at the 5-HT(1B) receptor causes TCA disorganization in rat barrel field cortex in the absence of elevated 5-HT.