Stretch-induced expression of vascular endothelial growth factor (VEGF) is thought to be important in mediating the exacerbation of diabetic retinopathy and other retinal neovascular diseases by systemic hypertension. However, the mechanisms underlying stretch-induced VEGF expression are not fully understood. We present novel findings demonstrating that stretch-induced VEGF expression in retinal capillary pericytes is mediated by PI3 kinase and PKC- but is not mediated by ERK1/2, classical/novel isoforms of PKC, Akt or Ras despite their activation by stretch. Cardiac-profile cyclic stretch at 60cpm increased VEGF mRNA expression in a time- and magnitude-dependent manner without altering mRNA stability. Stretch increased ERK1/2 phosphorylation, PI3 kinase activity, Akt phosphorylation and PKC- activity. Signaling pathways were explored using multiple pharmacologic inhibitors of PKC, MEK1,2 & PI3 kinase, adenovirus-mediated overexpression of ERK, PKC-, PKC-, PKC-, & Akt and dominant negative mutants of ERK, PKC-, Ras, PI3 kinase and Akt. Although stretch activated ERK1/2 through a Ras and PKC classical/novel isoform-dependent pathway, these pathways were not responsible for stretch-induced VEGF expression. Overexpression of dominant negative ERK and Ras had no effect on VEGF expression in these cells. In contrast, dominant negative PI3 kinase as well as pharmacologic inhibitors of PI3 kinase, blocked stretch-induced VEGF expression. Although stretch-induced PI3 kinase activation increased both Akt phosphorylation and activity of the atypical isoform PKC-, VEGF expression was dependent on PKC- but not Akt. In addition, PKC- did not mediate stretch-induced ERK 1/2 activation. These results suggest that stretch-induced expression of VEGF involves a novel mechanism dependent upon PI3 kinase mediated activation of PKC- that is independent of stretch-induced activation of ERK 1/2, classical/novel PKC isoforms, Ras or Akt. This mechanism may play a role in the well-documented association of concomitant hypertension with clinical exacerbation of neovascularization and vascular permeability.