Infrared-guided laser stimulation was used to examine the synaptic connectivity of neurons in rat barrel cortex. Layer V pyramidal neurons were visualized by infrared videomicroscopy and their membrane potential was recorded with patch pipettes. Presumptive presynaptic neurons were activated by uncaging glutamate with the light of a uv laser directed onto these neurons superfused with medium containing caged glutamate. Synaptic connections were identified by postsynaptic potentials following laser stimulation. The most frequent synaptic connections were found between layer V pyramidal neurons. The probability of this intralaminar input declined monotonically with the lateral distance between stimulated and recorded neuron. In contrast, input from layer II/III onto lamina V neurons showed a periodic organization. Synaptic connections originating from this lamina clearly reflected the barrel structure, with more input originating from the barrel column side, and less input from the barrel column centre. Thus, a barrel-specific organization seems to be especially pronounced for synaptic input from layer II/III to neurons of layer V.