1. To test the hypothesis that physiologically and morphologically different cortical nonpyramidal cells express different calcium-binding proteins, whole-cell current-clamp recording in vitro was combined with intracellular staining and double immunofluorescence in layer V of frontal cortex of rats 16-20 days old. 2. Nonpyramidal cells were first characterized as fast-spiking (FS) or low-threshold spike (LTS) cells, injected with biocytin, and subsequently stained immunohistochemically for parvalbumin and calbindinD28k. 3. FS cells were identified by input resistances < 350 M omega, spike width at half amplitude < 0.8 ms, and virtually no spike frequency adaptation of spike trains by depolarizing pulses. LTS cells were identified by input resistances > 350 M omega, spike width at half amplitude > 0.8 ms, and the discharge of low-threshold spikes from hyperpolarized potentials. Repetitive firing could be induced by a combination of stimulation-induced excitatory postsynaptic potentials with depolarization in FS cells. Repetitive firing was not observed in LTS cells under these conditions. 4. After biocytin injection of layer V cells characterized in this way, subsequent double immunostaining showed that all biocytin-labeled parvalbumin-immunoreactive cells (n = 18) belonged to the FS cells (FS-PV cells), whereas all biocytin-labeled calbindinD28k-immunoreactive cells (n = 10) belonged to the LTS cells (LTS-Calb cells). 5. FS-PV cells had smooth or sparsely spiny dendrites, whereas LTS-Calb cells had dendrites with a modest number of spines but fewer than pyramidal cells. FS-PV cells showed denser axonal branches near their somata and extended axons in a more horizontal direction. Some of them could be identified as basket cells by the presence of terminal boutons surrounding somata of other cells. LTS-Calb cells extended their main axons more vertically up to layer I. 6. Double immunofluorescent staining revealed that very few cells in layer V showed immunoreactivity for both calcium-binding proteins but that most cells immunoreactive for the calcium-binding proteins in layer V were also immunoreactive for gamma-aminobutyric acid. 7. These results suggest that GABAergic nonpyramidal cells in layer V of neocortex can be divided into two functional groups on the basis of different firing modes, axonal distributions, and calcium-binding protein immunoreactivity: 1) FS-PV cells show repetitive firing by synaptic activation, have axonal arborizations that are more dense near their somata and oriented horizontally, and the cells exhibit parvalbumin immunoreactivity and 2) LTS cells show low-threshold spikes, have more vertical axonal arborizations up to layer I, and exhibit calbindinD28K immunoreactivity.