Migration of dopamine (DA)-containing neurons and its guiding cues were histologically examined in the embryonic mesencephalon of normal mice. Cells immunoreactive (ir) for tyrosine hydroxylase (TH), a DA-synthesizing enzyme, were first detected on embryonic day 10 (E10) in the medio-basal part of the mesencephalon and were distributed throughout the entire length of the ventral mesencephalic wall at E12. By E14, TH-ir cells were located laterally along the ventral pial surface to form the primordia of the substantia nigra. Experiments with a single injection of bromodeoxyuridine, a thymidine analog, demonstrated that cells generated in the ventricular surface of the ventral mesencephalon at E11 migrated ventrally and then moved laterally to form the substantia nigra and the ventral tegmental area. Electron microscopic examination of the ventral mesencephalon of E12 mice disclosed that in the dorsal part ventrally migrating immature neurons made close contacts with the processes of radial glial cells. The expression of tenascin was transiently seen on radial glial processes between E10 and E13 coincident with the period of the ventral migration of mesencephalic DA neurons. By double immunostaining of E13 mesencephalon, ventrally migrating TH-ir cells were seen to be apposed to tenascin-bearing radial glial processes. On the other hand, laterally migrating neurons in the basal part of the mesencephalon were observed by electron microscopy to contact with tangentially arranged nerve fibers which were immunopositive for the 160 kDa neurofilament polypeptide at the light microscopic level from E10. Double immunostaining of E13 mesencephalon demonstrated that laterally migrating TH-ir cells were intermingled among neurofilament-ir fiber bundles. The cells of origin of the tangential nerve fibers were detected in the lateral part of the mesencephalon, when a fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) was injected into the basal part of the mesencephalon of fixed E12 mice. The present results suggest that guiding cues of the radial migration of mesencephalic DA neurons represent processes of radial glial cells which express tenascin. On the other hand, tangentially arranged nerve fibers originating from the lateral part of the mesencephalon may provide a scaffolding along which the mesencephalic DA neurons subsequently migrate laterally to form the ventral tegmental area and the substantia nigra.