The contribution of endoplasmic reticulum (ER) and phosphorylation of phospholamban (PLB) to the relaxant effect of cGMP- and cAMP-elevating agents was studied in feline aorta. Sodium nitroprusside (NP, 100 µM) completely relaxed contracture induced by 10 µM norepinephrine. This NP-induced relaxation was partially prevented by tetraethylammonium, suggesting that a fraction of NP-induced relaxation was mediated by activation of K⁺ channels. In the absence and presence of tetraethylammonium, the relaxant effect of NP was associated with a significant increase in Ser¹⁶ phosphorylation of PLB immunodetected by phosphorylation site-specific antibodies. The relaxant effect of NP on aortic strips precontracted with 80 mM KCl was significantly reduced by 1 µM thapsigargin. This decrease, which represents the ER contribution to the relaxant effect of NP, reached 23 ± 9% at 100 µM NP and was closely associated with a dose-dependent increase in Ser¹⁶ phosphorylation (128 ± 49% over control at 100 µM NP). Effects of NP were associated with a significant increase in activity of protein kinase G and were mimicked by 8-bromo-cGMP. Forskolin produced a dose-dependent relaxant effect on KCl-induced contracture, which reached 64 ± 8% at 50 µM and was associated with an increase in phosphorylation of Ser¹⁶ residue of PLB (88 ± 18% over control). Thapsigargin reduced this relaxant effect by 38 ± 9%. 8-Bromo-cAMP mimicked effects of forskolin. The ER-mediated relaxant effect and the increase in Ser¹⁶ phosphorylation produced by forskolin were partially blocked by the protein kinase A inhibitor H-89 (5 µM). The results indicate that ER partially contributes to the relaxant effect of NP and forskolin in feline aorta. This effect may be mediated by the associated increase in Ser¹⁶ phosphorylation of PLB.