For several decades, clinical and experimental observations suggested a relationship between steroids and glaucoma; however, the possibility that androgens are also involved in the glaucomatous changes in the optic nerve heads (ONH) has not been explored. Our previous findings that glaucomatous ONH astrocytes synthesize androgen-metabolising enzymes and overproduce a neuroactive androgen, 5alpha-androstane-3alpha, 17beta-diol (3alpha-diol) led us to propose that ONH astrocytes are androgen target cells. Androgens modulate different cellular processes through androgen receptor (AR). NFkB is a transcription factor that positively regulates AR transcription. Here, we analysed AR and NFkB expression in normal and glaucomatous ONH astrocytes in vitro, and in vivo in a monkey model of experimental glaucoma (ExpG) by quantitative real time RT-PCR, Western blotting and immunohistochemistry. We demonstrated that in vitro human glaucomatous ONH astrocytes express AR mRNA and protein at higher levels than normal astrocytes and that in vivo ONH astrocytes from eyes with ExpG showed increased nuclear and cytoplasmic AR immunostaining compared to control eyes. In the retina, retinal ganglion cells (RGC) demonstrated cytoplasmic staining both in control and in ExpG eyes. NFkB mRNA expression was higher in glaucomatous ONH astrocytes than in normal and more nuclear NFkB protein was detected in glaucomatous ONH astrocytes. In vivo immunopositive NFkB nuclear staining of ONH astrocytes in ONH and in RGC in retina was detected both in control and in ExpG eyes. We conclude that in addition to our published data, increase of AR and NFkB expression in glaucomatous ONH astrocytes provides strong evidence that androgens play a significant role in the pathophysiology of glaucoma.