The microbial polysaccharide HS-142-1 has been documented as an antagonist of natriuretic peptides. It inhibits activation and peptide binding to both guanylate receptors natriuretic peptide receptor (NPR)-A and NPR-B, but has no effect on the non-cyclase receptor NPR-C. At first sight the effect of HS-142-1 on peptide binding appears to be surmountable, suggesting that it might be competitive despite its chemically divergent nature. We explored its mode of action on wild-type NPR-A (WT), on a disulphide-bridged constitutively active mutant (C423S) and on truncated mutants lacking either their cytoplasmic domain (ΔKC) or both the cytoplasmic and the transmembrane domains (ECD). On the WT, HS-142-1 inhibited atrial natriuretic peptide (ANP) binding with a pK value of 6.51±0.07 (Kd = 0.31μM). It displayed a similar effect on the C423S mutant (pK = 6.31±0.11), indicating that its action might not be due to interference with receptor dimerization. HS-142-1 also inhibited ANP binding to ΔKC with a pK of 7.05±0.05 (Kd = 0.089μM), but it was inactive on ANP binding to ECD at a concentration of 10−4M, suggesting that the antagonism was not competitive at the peptide-binding site located on the ECD and that the transmembrane domain might be required. HS-142-1 also enhanced dissociation of NPR-A-bound 125I-ANP in the presence of excess unlabelled ANP, implying an allotopic (allosteric) mode of action for the antagonist.