The vasorelaxing properties of chocolate and wine might relate to the presence of phenolic compounds. One of the potential mechanisms involved is stimulation of endothelial nitric oxide (NO) production, as NO is a major regulator of vasodilatation. This study aimed to develop an in vitro assay using the hybrid human endothelial cell line EA.hy926 to rapidly screen phenolic compounds for their NO-stimulating potential. The assay was optimized, and a selection of 33 phenolics, namely, procyanidins, monomeric flavan-3-ols, flavonols, a flavone, a flavanone, a chalcone, a stilbene, and phenolic acids, was tested for their ability to enhance endothelial NO level. Resveratrol, a well-known enhancer of NO level, was included as a positive control. Of the 33 phenolics tested, only resveratrol (285% increase in NO level), quercetin (110% increase), epicatechingallate (ECg) (85% increase), and epigallocatechingallate (EGCg) (60% increase) were significant (P <or= 0.05) enhancers. Procyanidins showed a nonsignificant tendency to elevate NO level. Concentration-dependent correlations between enhanced NO level and endothelial nitric oxide synthase (eNOS) expression were demonstrated for the three polyphenols tested (resveratrol, ECg, and EGCg). Thus, an easy screening tool for change in cellular NO level was developed. Use of this assay showed that only a limited number of phenolic compounds might enhance NO level with an increased amount of eNOS enzyme as a possible contributing mechanism.