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This study evaluated the distribution of goblet cells and the expression of MUC5AC mRNA in the canine nictitating membrane. The distribution of goblet cells in the nictitating membrane and temporal bulbar conjunctiva of beagle dogs was examined by histochemical analysis of impression cytology specimens and frozen sections. MUC5AC mRNA was detected by the reverse transcription polymerase chain reaction (RT-PCR). The distribution of MUC5AC mRNA was also examined by in situ hybridization using digoxigenin-labeled antisense and sense RNA probes. Histochemical analysis showed that the canine nictitating membrane epithelium contained many more periodic acid-Schiff positive goblet cells, particularly on the palpebral side, compared with the temporal bulbar conjunctiva. RT-PCR revealed that MUC5AC was expressed in both the nictitating membrane and in conjunctival tissue. When the distribution of MUC5AC mRNA was assessed by in situ hybridization, its expression was high on the palpebral side of the nictitating membrane and low in the temporal bulbar conjunctiva. MUC5AC mRNA expression corresponded with the distribution of goblet cells by histochemical examination. In conclusion, there were numerous goblet cells in the canine nictitating membrane epithelium, particularly on the palpebral side, and MUC5AC mRNA was expressed in the nictitating membrane epithelium at locations corresponding to the goblet cells.