1. In the present study we tested the effect of arg-gly-asp (RGD) peptides on vasomotor responses in rat isolated mesenteric arteries. More specifically, the hypothesis was tested that RGD interaction with integrins mediates relaxation attributed to endothelium-derived hyperpolarizing factor (EDHF). 2. The presence of the beta3 integrin subunit was shown by western blot analysis. To study its functional role, arteries (355 +/- 11 microm; n = 50) were mounted in a wire myograph set-up to measure isometric force generation. After blockade of nitric oxide synthesis with N(G)-nitro-L-arginine (0.1 mmol/L) and prostaglandin synthesis with indomethacin (10 micromol/L), methacholine (10 micromol/L) induced a transient relaxation within 1 min of 72 +/- 4.0% (as percentage of precontraction with phenylephrine; n = 27). 3. These responses were inhibited by a 60 mmol/L potassium buffer (18 +/- 6.0%; n = 6) or endothelium denudation (12 +/- 3.2%; n = 7), consistent with EDHF. 4. A function-blocking monoclonal antibody against the integrin beta3 chain did not affect relaxation. 5. The RGD peptides gly-arg-gly-asp-thr-pro (GRGDTP), gly-arg-gly-asp-ser (GRGDS) and cyclic RGD, ligands for the RGD binding site of integrins, also did not affect relaxation induced by methacholine. 6. Cyclic RGD increased contraction from 91 +/- 3 to 98 +/- 3% (as percentage of 120 mmol/L potassium). 7. In conclusion, these data show that vasomotor responses related to integrins are small and not involved in hyperpolarization attributed to EDHF in rat mesenteric artery.