Little is known about how neurons in the different layers of the mammalian cerebral cortex are specified at the molecular level. Expression of two homologues of the Drosophila homeobox Cut gene, Cux-1 and Cux-2, is strikingly specific to the pyramidal neurons of the upper layers (II–IV) of the murine cortex, suggesting that they may define the molecular identity of these neurons. An antibody against Cux-1 labels the nucleus of most of the postmitotic upper layer neurons but does not label parvoalbumin-positive cortical interneurons that derive from the medial ganglionic eminence. Cux-1 and Cux-2 represent early markers of neuronal differentiation; both genes are expressed in postmitotic cortical neurons from embryonic stages to adulthood and in the proliferative regions of the developing cortex. In precursors cells, Cux-1 immunoreactivity is weak and diffuse in the cytoplasm and nucleus of ventricular zone (VZ) cells, whereas it is nuclear in the majority of bromodeoxyuridine (BrdU)-positive subventricular zone (SVZ) dividing cells, suggesting that Cux-1 function is first activated in SVZ cells. Cux-2 mRNA expression is also found in the embryonic SVZ, overlapping with BrdU-positive dividing precursors, but it is not expressed in the VZ. A null mutation in Pax-6 disrupts Cux-2 expression in the SVZ and Cux-1 and Cux-2 expression in the postmigratory cortical neurons. Thus, these data support the existence of an intermediate neuronal precursor in the SVZ dedicated to the generation of upper layer neurons, marked specifically by Cux-2. The patterns of expression of Cux genes suggest potential roles as determinants of the neuronal fate of the upper cortical layer neurons. J. Comp. Neurol. 479:168–180, 2004. © 2004 Wiley-Liss, Inc.