In protriptyline (25 mg/kg) pretreated rats stereotactic 5,7-dihydroxytryptamine (5,7-DHT) lesions of the medial plus laternal 5-hydroxytryptamine (5-HE) bundles in the mesencephalon increased the 5-HT fluorescence in these bundles, and reduced the in vitro uptake of [3H] 5-HT in the hypothalamus to 16% of control values after 2 mug 5,7-DHT/4mul and 12% after 4 mug 5,7-DHT/4mul, and in the cortex cerebri to 35 and 34% of control values, respectively. Selective lesion of the medial 5-HT bundle reduced [3H] 5-HT uptake both in hypothalamus and in cortex cerebri to 45-48% of control values, while selective lesion of the lateral 5-HT bundles significantly reduced [3H] 5-HT uptake only in cortex (to 73-75%). No significant change was observed in [3H] noradreanaline uptake after any injection, or in [3H] 5-HT uptake after vehicle injections. Locomotor activity in an open field 3-10 days postoperatively was significantly reduced by lesions of the medial plus lateral 5-HT bundles. 5-Hdroxytryptophan (50 mg/kg) and a peripheral decarboxylase inhibitor (MK 486, 75 mg/kg) 17 days postoperatively induced a pronounced behavioral "5-HT syndrome" in these rats with medial plus lateral lesions but not in controls. Pain sensitivity, as measured by the hot plate test, was not changed by any lesion, even when tryptophan hydroxylase was partly inhibited with alpha-propyldopacetamide (100 mg/kg). Morphine analgesia and acquisition of a one-way avoidance response also were unchanged. Apomorphine (2 mg/kg)-induced locomotor activity and stereotyped behavior, as measured in an Animex activity meter, were not significantly different from control values in the 5,7-DHT groups. It was concluded that the medial 5-JT BUNDLE INNERVATES BOTH THE HYPOTHALAMUS AND THE CORTEX CEREBRI AND THE LATERAL 5-HT bundle mainly the cortex. These ascending 5-HT neurons are involved in maintaining open field ambulation. No wupport was obtained for the view that they are involved in pain mechanisms, in morphine-induced analgesia, in apomorphine-induced motor behavior, or in one-way avoidance learning.