Recently, two orthologues of the Drosophila homeobox Cut gene, Cux-1 and Cux-2, have been identified as restricted molecular markers of upper layer (II–IV) neurons in the murine cerebral cortex. We show that during early postnatal life, from P0 to P10, Cux-1 and Cux-2 mRNA are coexpressed in all primary sensory cortices. Antisera to Cux-1 and Cux-2 immunoreactivities preferentially label neurons in the barrel walls of the primary somatosensory cortex (S1). Subsequently, Cux-1 remains enriched in sensory cortices, whereas Cux-2 expression enlarges to comprise the frontal and insular areas. The laminar distribution of Cux-1 and Cux-2 differs: Cux-1 follows a layer IV to layer II decreasing gradient of expression, whereas Cux-2 expression is homogeneous across layers IV–II. No colocalization was found with GABA and birth dating experiments showed that Cux-1–positive neurons in layer IV are born during a restricted period, E13.5–E14.5, suggesting that Cux-1 is a useful molecular marker of the glutamatergic neurons of layer IV. We examined Cux-1 and Cux-2 in barrel-defective mouse strains, the VMAT2 KO, the MAOA KO, and the Adcyl 1brl strain. A normal expression level of Cux-1 and Cux-2 was found in layer IV, despite the lack of segregation of the neurons as barrels. Conversely, in Reeler mice, Cux-1 and Cux-2 had a distinct laminar distribution: the Cux-1–positive neurons had an inverted deep localization, whereas the Cux-2–positive neurons were distributed throughout the cortical thickness, suggesting that Cux-2 expression is more widely expressed in the inverted cortex of reeler mutants. Our results indicate that Cux-1 is a useful marker of the layer IV neurons in S1, and that Cux-1 and Cux-2 are differently regulated in the upper layers of the cerebral cortex. © 2006 Wiley-Liss, Inc.