The goal of the present study was to investigate the pattern of maturation of callosal projecting neurons in a well-studied mammalian visual system with unique structural and functional properties. Studies of the distribution pattern of interhemispheric connections in the adult tree shrew primary visual cortex reveal not only a high concentration of labeled neurons along the area 17/18 border, as in standard experimental animals such as the cat and monkey, but also numerous callosal projecting neurons in the adjacent dorsal part of area 17, which largely corresponds to the binocular visual field (Kretz and Rager, Exp. Brain Res. 82:271, '90). Callosal projections were anatomically traced in 11 tree shrews (Tupaia belangeri) at various ages between postnatal day 7 (7, 9, 10, 13, 15, 17, 19, and 26 days old) and adulthood (107 days old). In each animal, four injections of wheat germ agglutinin conjugated to horseradish peroxidase were made in a standard configuration into the striate cortex of one hemisphere. In young tree shrews only 7 and 9 days old, heavily labeled terminal axon structures could be seen in the white matter and in layer VI of the opposite hemisphere. Only a few labeled neurons, however, were detected in layer III. The small number of labeled neurons indicated that early in postnatal development, only a few callosal axons had invaded the upper cortical layers. By 10 days of age, the number of supragranular neurons was increasing and the maximal value was counted in a 13-day-old tree shrew. A sharp decline in the number of labeled supragranular neurons was noticed--about 94% in our case--between days 13 and 15. In animals more than 15 days old, the distribution pattern and the density of the neurons looked like the pattern seen in the adult Tupaia brain. The labeled cells were mostly concentrated in layers II and III. The majority of neurons resembled typical pyramidal cells. However, some of the neurons in sublayer IIIc had elongated cell bodies oriented parallel to the laminar boundaries. In contrast to the supragranular cells found in all stages investigated, small populations of labeled cells in layer VI were observed in 9- to 17-day-old tree shrews only. In young postnatal animals 7 to 13 days old, a peculiar cell type was labeled on the ipsilateral side. In coronal sections these cell bodies formed a continuous band that extended from the ventricular wall to the subcortical white matter. These cells might belong to a population of cells still in migration.