We investigated the effects of the sulfhydryl-donor, N-acetylcysteine (NAC), on nitroglycerin (NTG)-induced relaxation of the vascular smooth muscle. Addition of histamine to isolated porcine coronary arteries induced an initial rapid contraction followed by a gradual decrease in tonic contraction. NTG applied to the coronary artery strips before histamine caused relaxation of the histamine-induced rapid (3 min) and tonic (48 min) contraction. The inhibition of the tonic contraction by NTG was less at 48 min than at 3 min. Application of NAC (NTG-NAC) enhanced the relaxing effects of NTG on the histamine-induced tonic contraction rather than the acute contraction. In phosphorylation studies, changes in the phosphorylation of an intermediate filament, desmin, were parallel with changes in contraction in NTG-treated and NTG-NAC samples at 48 min. These phosphorylation changes of desmin at 48 min, which might be responsible for tonic phase contraction, were more extensive than those of myosin light chain (MLC) phosphorylation at 3 min, which might be responsible for acute contraction. These results suggest that treatment with the sulfhydryl donor, NAC, inhibited the phosphorylation of desmin associated with the enhancement of NTG-induced relaxation, which might be related to the mechanisms of recovery from NTG tolerance by sulfhydryl groups.