The sarcoplasmic reticulum-bound creatine kinase from rabbit skeletal muscle was inhibited by the nitric oxide donor S-nitrosoglutathione (GSNO). This led to a decrease in Ca2+ uptake in sarcoplasmic reticulum vesicles when the transport was driven by ATP generated from phosphocreatine and ADP. In contrast, the Ca 2+ transport measured using 2 mM ATP as substrate was unaffected by GSNO up to 200 microM. GSNO (5-20 microM) inhibited the activity of both soluble and membrane-bound creatine kinase. Oxyhemoglobin (15-40 microM) protected creatine kinase against inactivation by GSNO. The inhibition by 10 microM GSNO was reversed by the addition of dithiothreitol (2 mM). The results indicate that nitric oxide (NO, including NO+, NO and NO-) inactivates creatine kinase in vitro by promoting nitrosylation of critical sulphydryl groups of the enzyme.