We have previously shown that ANP causes differential constriction of the splenic vasculature of the rat (veins greater than arteries), which may be inhibited by blocking the production of cGMP with A7195. In this paper, we report experiments done on vessels derived from guanylyl cyclase (GC)-A knockout mice. Small splenic arteries (~150-µm diameter) and veins (~250-µm diameter) were dissected from male GC-A-deficient 129sv mice or age-matched wild-type controls and mounted in a wire myograph. In the wild-type mice, ANP exhibited higher potency in the veins than in the arteries (EC₅₀ values wild-type mice: artery, 8 ± 3 × 10⁹ M, n = 5 vs. vein, 6 ± 4 × 10¹⁰ M, n = 5; P < 0.05). The concentration-response curve for ANP-induced vasoconstriction was also shifted leftward in denuded compared with intact arteries (EC₅₀ values: denuded artery: 5 ± 3 × 10¹⁰ M, n = 5 vs. intact artery, 8 ± 3 × 10⁹ M, n = 5; P < 0.05), i.e., the denuded vessels were more reactive. By contrast, ANP caused no significant change in tension from baseline in intact splenic arteries, intact splenic veins, or denuded splenic arteries derived from the GC-A-deficient mice, although these vessels did show normal concentration-dependent increases in tension to phenylephrine. We conclude that ANP causes vasoconstriction in the splenic vasculature by an endothelium-independent mechanism, mediated via guanylyl cyclase.