There is clinical and experimental evidence that elevated intraocular pressure (IOP), a mechanical stress, is involved in the pathogenesis of glaucomatous optic neuropathy. The mechanism by which astrocytes in the optic nerve head (ONH) respond to changes in IOP is under study. Gene transcription by ONH astrocytes exposed either to 60 mmHg hydrostatic pressure (HP) or control ambient pressure (CP) for 6, 24, and 48 h was compared using Affymetrix GeneChip microarrays to identify HP-responsive genes. Data were normalized across arrays within each gene. A linear regression model applied to test effect of time and HP on changes in expression level identified 596 genes affected by HP over time. Using GeneSpring analysis we selected genes whose average expression level increased or decreased more than 1.5-fold at 6, 24, or 48 h. Expression of selected genes was confirmed by real-time RT-PCR; protein levels were detected by Western blot. Among the genes highly responsive to HP were those involved in signal transduction, such as Rho nucleotide exchange factors, Ras p21 protein activator, tyrosine kinases and serine threonine kinases, and genes involved in transcriptional regulation, such as c-Fos, Egr2, and Smad3. Other genes that increased expression included ATP-binding cassettes, solute carriers, and genes associated with lipid metabolism. Among the genes that decreased expression under HP were genes encoding for dual activity phosphatases, transcription factors, and enzymes involved in protein degradation. These HP-responsive genes may be important in the establishment and maintenance of the ONH astrocyte phenotype under conditions of elevated IOP in glaucoma.