The callosal projection is traditionally believed to be exclusively excitatory in function. A new method combining lectin-conjugated colloidal gold for retrograde tracing with immunofluorescence for GABA was used to investigate this question. The large majority of neurons retrogradely labeled after injections of tracer into contralateral S-I were GABA-negative cells in layers II-VI. However, GABA-positive neurons projecting to contralateral S-I were also seen. The majority of double-labeled cells were only weakly labeled with tracer; they were located both in superficial and deep cortical layers and represented at least 1% of all retrogradely labeled neurons. Neurons double-labeled for both tracer and GABA in upper layers were found mainly within the central patch of callosally projecting neurons, whereas those in deep layers were scattered throughout contralateral cortex.