Coronary endothelial luminal membrane (CELM) oligosaccharides (O) and lectins (L) are involved in flow detection. Flow may induce a reversible L‐O interaction causing flow‐induced endothelial (FIE) cardiac effects. Because N‐Acetylglucosamine (GlcNac) is a component of glycocalyx O, we isolated CELM GlcNac‐binding L (using GlcNac‐affinity probe) and show their role in cardiac and vascular FIE responses. The GlcNac‐affinity probe is a 460 kDa GlcNac polymer (GlcNac‐Pol). In the heart: 1) Intracoronary given GlcNac‐Pol upon binding to CELM, reduces the FIE inotropic and dromotropic effects. 2) GlcNac‐Pol used as an affinity probe isolated CELM GlcNac‐recognizing L; 35 individual L were identified, some of which likely participate in FIE and in GlcNac‐Pol‐induced effects. 3) In isolated blood vessels perfuse at controlled flow rates; FIE vasodilatation (FIEV) is blocked by: binding GlcNac‐Pol to CELM, Hyaluronidase, L‐NAME plus Indomethacin and Amiloride; a ENaC blocker. We also show ENac is present in CELM and is a L. These suggest FIEV is due to: a) FIE ENaC‐hyaluronidate (a GlcNac polymer) interaction, b) release of NO and prostaglandins and c) GlcNac‐Pol upon binding to ENaC inhibits release of paracrine mediators. Funded by CONACyT SEP‐42567, CONACyT‐SALUD 2004‐C01‐156.