Events underlying derepression of the nitrogen fixation (nif) genes in Klebsiella pneumoniae M5A1 were analyzed in vivo by comparing the effects of selective inhibitors of transcription and translation on subsequent nitrogenase activity (rate of acetylene reduction). When batch cultures were induced for derepression, an 87-min lag separated ammonium ion/oxygen removal and the appearance of activity. To prevent eventual activity by adding inhibitors during this period, it was found necessary to add rifampin, ammonium ion, or chloramphenicol more than 50, 20, or 10 min, respectively, before activity appeared in a parallel control. When these inhibitors were added to cultures in which nitrogenase activity had already appeared, further increase in activity was not stopped by rifampin or ammonium ion until 45 or 20 min, respectively, after addition. Chloramphenicol stopped further increase in nitrogenase activity almost immediately. These data indicated a nif operon whose transcription/translation consumes 40 min. When the kinetics of β-galactosidase induction were analyzed under similar conditions, it was found that 7 min separated the initiation of transcription and the first completions of translation. Extrapolating from this, we find the nif operon to occupy 17 average gene lengths. It is argued that the nif structural genes in K. pneumoniae are contained on one operon, and further, that the disparity between the kinetics of inhibition by rifampin and those by ammonium ion suggests regulation at a locus other than the operator.