Tamm‐Horsfall protein (THP), also called uromodulin, is an 86kD acidic, single‐chain, glycoprotein produced in the kidney and excreted into the urine. THP binds various molecules of the immune system including immunoglobulins (Ig), complement 1q (C1q), and factor H (fH). Heparin is a negatively‐charged, sulfated polysaccharide that binds to a wide range of proteins, including C1q, fH, antithrombin III (ATIII), basic fibroblast growth factor (FGF2), and interleukin‐10 (IL‐10). The purpose of this study was to determine if heparin competed with THP for binding to proteins that interact with both THP and heparin (i.e. C1q and fH), and to screen other heparin‐binding proteins for previously unidentified interactions with THP. Dot blots were performed by immobilizing 1 μg of C1q, fH, and bovine serum albumin (BSA) to 4 strips of nitrocellulose (NC). One NC strip was stained with amido black and the other 3 NC strips were incubated with either THP (10 μg/ml), THP (10 μg/ml)/heparin (20 μg/ml) (sodium salt from porcine intestine), or no THP/heparin, before incubation with sheep anti‐THP antiserum and then donkey anti‐sheep IgG ‐ HRP. These blots showed that THP bound strongly to C1q and fH, and that this binding was inhibited by heparin. Heparin also inhibited the background binding of the antisera to C1q. ELISAs were used to quantify the ability of heparin to inhibit C1q (5 μg/ml) binding to immobilized THP (40 μg/ml). A steep inhibition curve was obtained, with no inhibition of C1q binding to immobilized THP occurring with 0.02 units/ml heparin, 50% inhibition of binding occurring with between 0.04 and 0.05 units heparin/ml, and 90% inhibition occurring with 0.1 unit heparin/ml (170 units/mg heparin). Control wells showed that heparin's inhibition of C1q binding to THP was not due to inhibition by heparin of the antisera's binding to C1q. Additionally, dot blots were used to evaluate binding of soluble THP to ATIII, IL‐10, and FGF2, three of the many proteins that heparin has been shown to bind. These blots demonstrated that THP bound to FGF2 and IL‐10, and this interaction was blocked by the addition of heparin. THP did not bind ATIII. In summary, heparin inhibited THP's binding to NC‐immobilized C1q and fH, as well as inhibiting immobilized THP's binding to soluble C1q in ELISAs. Protein‐protein interactions between THP and FGF2 and IL‐10, that were sensitive to heparin inhibition, were discovered. Heparin and THP coexist in the urine, especially after intravenous heparin administration or when heparin is instilled into the bladder as a therapy for interstitial nephritis. Mast cells are prominent in the wall of the urinary bladder in interstitial cystitis, so competition of heparin and THP for binding to other proteins may be important in this disease. Further studies to assess the physiological significance in THP/heparin competition for binding to various proteins is warranted.Support or Funding InformationFunded by the PNWU Research Seed Program.