Layer VI is the origin of the massive feedback connection from the cortex to the thalamus, yet its complement of cell types and their connections is poorly understood. The physiological and morphological properties of corticofugal neurons of layer VI of mouse primary visual cortex were investigated in slices loaded with the Ca(2+) indicator fura-2AM. To identify corticofugal neurons, electrical stimulation of the white matter (WM) was done in conjunction with calcium imaging to detect neurons that responded with changes in intracellular Ca(2+) concentrations in response to the stimulation. Subsequent whole cell recordings confirmed that they discharged antidromic action potentials after WM stimulation. Antidromically activated neurons were more excitable and had different spiking properties than neighboring nonantidromic neurons, although both groups had similar input resistances. Furthermore, antidromic neurons possessed narrower action potentials and smaller afterhyperpolarizations. Additionally, three-dimensional reconstructions indicated that antidromically activated neurons had a distinct morphology with longer apical dendrites and fewer nonprimary dendrites than nonantidromic cells. To identify the antidromic neurons, rhodamine microspheres were injected into the dorsal lateral geniculate nucleus of the thalamus and allowed to retrogradely transport back to the somata of the layer VI cortico-geniculate neurons. Physiological and anatomical analysis indicated that most antidromic neurons were likely to be cortico-geniculate neurons. Our results show that cortico-thalamic neurons represent a specific functional and morphological class of layer VI neurons.