Layer I is a dense synaptic zone ubiquitous in cerebral cortex. Here we describe a novel in vitro preparation of rat somatosensory (SI) neocortical slices that isolates the fibers that extend horizontally through layer I, and allows intracellular and extracellular analysis of synaptic input to dendrites in layer I. Current source-density analysis of this isolated horizontal layer I input reveals monophasic current sinks restricted to layer I and the most superficial part of layer II. The layer I synaptic response of each neuron was correlated with its morphology by filling penetrated cells with biocytin. All filled cells that responded to horizontal layer I inputs were pyramidal neurons in layers II, III, or V with distal apical dendrites in layer I. There was no evidence of antidromic activation from isolated layer I stimulation, and HRP injected into layer I was not transported via the isolated layer I pathway to cortical neurons within the slice. Therefore, this preparation provides a unique way to study an extrinsic synaptic input localized to the most distal apical dendrites of many pyramidal neurons. In contrast to the EPSP-IPSP sequence characteristically evoked by deep layer stimulation, horizontal layer I inputs evoked long- lasting EPSPs (approximately 50 msec); IPSPs were observed only rarely, in the most superficial neurons. Horizontal layer I-evoked EPSPs were blocked by the non-NMDA glutamate receptor antagonist 6-cyano-7- nitroquinoxaline-2,3-dione. Consistent with the very distal site of layer I inputs to layer V pyramidal neurons, the amplitudes of initial EPSPs were insensitive to manipulations of the somatic membrane potential. However, these distal EPSPs were greatly attenuated when combined with IPSPs evoked by deep layer stimulation, indicating that the proximal inputs may modulate distal EPSPs with shunting inhibition. In many layer V neurons, the initial EPSP evoked by horizontal layer I stimulation was followed by a variable late depolarization that was blocked by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid. Since these late depolarizations were enhanced by somatic depolarization and abolished by hyperpolarization, they appeared to be generated postsynaptically at a site more proximal than the initial EPSP that was insensitive to these manipulations. Synaptic inputs to the distal tufts of pyramidal neurons may trigger active currents along the apical dendrites that amplify the EPSP on its way to the soma.