Double immunofluorescence histochemistry for calcium-binding proteins was performed in the caudal subnucleus of the rat spinal trigeminal nucleus; for calbindin D28k (CB) and calretinin (CR), for CB and parvalbumin (PV), and for CB and CR. CB-immunoreactive (-ir) neurons were seen 1.7 times more frequently than CR-ir neurons and 5.5 times more frequently than PV-ir neurons. About 70-90% of these neurons were distributed in substantia gelatinosa. Co-existence of CB and CR was indicated in 2.3% of CB-ir and 3.9% of CR-ir neurons. Co-existence of CB and PV was indicated in 1.0% of CB-ir and 5.5% of PV-ir neurons. Co-existence of CR and PV was indicated in 1.4% of CR-ir and 5.1% of PV-ir neurons. In these doubly immunostained neurons, 59.5-69.5% were observed in substantia gelatinosa, 5.9-17.8% in the marginal zone, and 12.7-31.0% in the magnocellular part.