Nitric oxide (NO) released from cells or other sources is very rapidly oxidized to nitrite in the presence of oxygen. This article describes a procedure utilizing 1,1′-dimethylferrocene as the reducing agent for nitrite to nitric oxide and its subsequent determination by chemiluminescence. The introduced method allows the determination of nitric oxide together with nitrite. The total nitric oxide content of a sample can be quantified with high reproducibility in the picomolar range. A new microreaction vessel facilitates the convenient introduction of samples and of the reducing solution. The reaction is fast at room temperature and thus requires no additional heating.