Theories of information processing and plasticity in mammalian cortex often rely on knowledge of intracortical networks studied in rodent cortex. Accordingly, the contribution of all cells involved in this circuitry is potentially significant, including connections from a subset of neurons that persist from the developmental subplate, called subgriseal neurons in the present study. Ascending corticocortical connections from subgriseal neurons were identified by using in vivo transport of fluorescent retrograde tracers from discrete applications confined to cortical layer I (approximately 1 mm2) or from injections placed into superficial cortical layers. Applications restricted to cortical layer I can be identified by a subsequent retrograde labeling pattern that includes neurons in layers II/III and V but not those in layer IV. In contrast, when retrograde tracer is deposited in layers II/III, layer IV cells are also labeled. By using this identification technique in juvenile and adult rats, widespread interareal projections to superficial layers, including unequivocal connections to cortical layer I, were found to originate from a tangential band of neurons directly below the conventionally identified gray matter (i.e., subgriseal) and from a smaller number of cells in the white matter (WM) proper. Subgriseal and WM neurons were labeled below application and injection sites in somatosensory, auditory, visual, motor, frontal, and adjacent areas at distances of more than 4 mm. However, the subgriseal-to-superficial pathway was not sensitive to nonfluorescent retrograde tracers including horseradish peroxidase. Because neurons in the deeper cortical layers can be strongly influenced through input to their apical dendritic extensions in cortical layer I, the widespread connections described in the present study indicate that the ascending subgriseal projections should be considered in models of mature cortical function.