This study examined the factors that may be involved in altering the function of pineal gland in hypoxic conditions. Adult Wistar rats were subjected to hypoxia and the pineal gland was examined for the mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), endothelial, neuronal and inducible nitric oxide synthase (eNOS, nNOS, iNOS) at 3 hr-14 days after hypoxic exposure by real time reverse transcription-polymerase chain reaction, Western blotting and immunohistochemistry. Upregulated mRNA and protein expression of HIF-1alpha, VEGF, eNOS, nNOS and iNOS was observed in response to hypoxia. VEGF concentrations as determined by enzyme immunoassay and nitric oxide (NO) production measured by colorimetric assay were significantly higher after hypoxic exposure when compared with the controls. Melatonin content of the pineal gland, as determined by ELISA, was significantly reduced after the hypoxic exposure. Dilated blood vessels expressing eNOS were observed in hypoxic rats. Cells immunoreactive for VEGF were identified as the astrocytes whereas those immunoreactive for iNOS were pinealocytes and macrophages. Our findings indicate that excess production of VEGF and NO in pineal gland in response to hypoxia may be involved in increased vascular permeability as evidenced by an enhanced leakage of rhodamine isothiocyanate (RhIC). The increased vascular permeability may allow free access of serum-derived substances in the pineal gland that may affect the secretory function of the pinealocytes. Administration of exogenous melatonin may be beneficial as it reduced VEGF concentration and NO production significantly in hypoxic rats, and leakage of RhIC was concomitantly reduced.