A new on‐line method for measuring acetylene reduction is described. It consists of a gas‐flow cell connected to an electronic gas‐mixing system and an automatic sample loop in the gas chromatograph. Alternatively, ethylene can be determined by using laser‐based trace gas detection. The laser‐based trace gas detection technique achieves a detection limit that is three orders of magnitude better than gas chromatography. We have applied the on‐line method to the measurement of nitrogen fixation in a culture of the heterocystous cyanobacterium Nodularia spumigena and compared it with conventional batch‐type incubations. Incubation of N. spumigena in the gas‐flow cell resulted in very short response times with a steady‐state flux of ethylene obtained within 2 min. Nitrogenase was shown to respond immediately to changes in light and oxygen. Monitoring of nitrogenase activity could be continued for several hours without having a negative impact on nitrogen fixation rates in N. spumigena. This was not the case in batch incubations, in which changes in nitrogenase activities were recorded during incubations, probably as a result of varying oxygen concentrations. It was therefore concluded that the on‐line method is superior to batch incubations when rates of nitrogenase activity are to be measured. The method is suitable for natural samples (water or sediment).