The hypothesis that the six cytoarchitectonically distinct subdivisions of the zona incerta send efferent projections to different parts of the brain was tested using retrograde fluorescent tracing techniques. Injections of tracer in the superior colliculus, pontomesencephalic tegmentum, thalamus or spinal cord resulted in characteristic and distinctive patterns of labelling in the zona incerta, suggesting that the different subdivisions of the zona incerta give rise to different sets of efferent projections. Thus, injections into the deep layers of the superior colliculus resulted in retrogradely labelled neurons predominantly in the zona incerta pars ventralis, whereas injections into the pontomesencephalic tegmentum, avoiding the superior colliculus, labelled neurons mostly in the zona incerta pars dorsalis. Parafascicular thalamic injections resulted in labelled neurons in the zona incerta pars rostropolaris, pars dorsalis, pars caudalis and a few in the pars ventralis. The spinal cord injections labelled predominantly the magnocellular cells of the zona incerta. The results of the double labelling experiment also give support to the conclusion that different areas of the zona incerta give rise to largely individual sets of efferent projections. Combined injections of tracers into the superior colliculus and thalamus resulted in the labelling of two essentially independent cell groups, the tectal projecting cells being found mainly in the zona incerta pars ventralis and the thalamic projecting cells being found in the zona incerta pars rostropolaris, pars dorsalis and pars caudalis. However, a small number of double labelled cells was found. These cells, which presumably send collateral projections to both the tectum and the thalamus, were found mainly in the dorsal part of the zona incerta pars ventralis, immediately next to the pars dorsalis. In the light of these findings it would appear that the zona incerta may be organised into largely distinct subdivisions, each of which has its own set of cytoarchitectonic and efferent projection characteristics.