Nitric oxide sensitive guanylyl cyclase (NOsGC), a hemoprotein and the major physiological receptor for nitric oxide (NO), is a heterodimer with the α1/β1 and α2/β1 isoforms known to be important for NO-signaling and conversion of GTP to cGMP in humans. Two innovative classes of compounds modulating the NO/cGMP signaling pathway have been discovered: the heme-dependent sGC stimulators, that stimulate NOsGC directly and also increase the affinity towards NO, and the heme-independent sGC activators, that are thought to bind to oxidized and heme-free NOsGC in tissues exposed to oxidative stress. In the current study, we evaluate the effects of the sGC activators BAY 58–2667 (cinaciguat) and BAY 60–2770 on the isoforms α1/β1 and α2/β1 expressed in Sf9 cells. Western blot analysis of cytosolic fractions revealed a decrease in overexpressed NOsGC in the presence of sGC activators, which is dependent on an intact catalytic site of the enzyme. For both isoforms, we show a higher efficacy for BAY 60–2770 compared to cinaciguat after purification of NOsGC by affinity and size exclusion chromatography. Using a new experimental strategy of expression of NOsGC with activator and subsequent purification, we demonstrate a stable insertion of activator drugs into the enzyme during protein biosynthesis independent of the heme redox state. We postulate that the balance between stable insertion of activator during de novo synthesis and replacement of NOsGC ferric heme in tissues exposed to oxidative stress can be influenced by the dosage regimen.