Soluble guanylyl cyclase (sGC) is a key enzyme of the ^•NO/cGMP pathway. Many cardiovascular disorders are associated with reduced ^•NO-mediated effects, while vascular superoxide (O₂^•−) production is increased. Both radicals rapidly react to peroxynitrite. We investigated whether peroxynitrite affects the activity and protein expression of sGC in intact vascular preparations. Catalytic sGC activity and expression of the sGC-β₁ subunit was measured by conversion of radiolabeled GTP and western blot, respectively, using cytosolic extracts from rat aorta that had been incubated for 4 h with NO/O₂^•− systems (devoid of free NO) generating either 0.13 μM or 7.5 μM peroxynitrite/min. Incubation of rat aorta with 0.13 μM peroxynitrite/min had no effect. In striking contrast, incubation with 7.5 μM peroxynitrite/min resulted in a shift of the concentration-response curve obtained with a NO donor (p = .0004) and a reduction of maximal specific activity from 3579 ± 495 to 2422 ± 265 pmol cGMP/mg/min (p = .036). The expression of the sGC-β₁ subunit was unchanged. Exposure of aorta to the O₂^•− component had no effect, while exposure to the NO-component reduced sGC expression to 58.8 ± 7% (p < .001) and maximal sGC activity from 4041 ± 992 to 1429 ± 491 pmol cGMP/mg/min (p = .031). These data suggest that continuous generation of extracellular peroxynitrite might interfere with the NO/cGMP signaling in vascular cells.