Renal and cardiac fibrosis leading to organ failure are complications of both diabetes and hypertension. These disease processes, when combined, exacerbate development of fibrotic complications. Control of latent transforming growth factor (TGF)-β activation is a potential determinant of fibrotic progression. Both glucose and angiotensin II (Ang II) upregulate thrombospondin-1 (TSP1), a major activator of latent TGF-β, and stimulate increased TGF-β activity. We previously showed that high glucose stimulated TSP1-dependent TGF-β activation in rat mesangial cells (RMCs). In this paper, we examined whether Ang II similarly upregulates TSP1 production and TSP1-dependent TGF-β activation alone or in combination with high glucose concentrations. Ang II and high glucose stimulated increases in TSP1 protein levels in the conditioned media of both rat cardiac fibroblasts (RCFs) and rat mesangial cells (RMCs). Meanwhile, Ang II stimulated increases in both TGF-β activity and protein by RMCs, whereas, RCFs responded to both Ang II and high glucose with increased TGF-β activity in the absence of altered TGF-β protein levels. A combination of Ang II and high glucose induced synergistic TGF-β activation by RCFs. Moreover, Ang II induction of TSP1 and increased TGF-β activity were blocked by losartan, an antagonist of the Ang II type 1 (AT1) receptor. The increase in TSP1 expression leads to increased TGF-β activity upon Ang II and/or glucose treatment, since peptide antagonists of TSP1-mediated TGF-β activation blocked Ang II and glucose-induced TGF-β activation. Our data support a role for TSP1 in the development and progression of renal and cardiac fibrosis in hypertension and diabetes.