Under 12/12 h light/dark cycles, 1 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, pH 8.0) added at the start of the dark period, inhibited the increase of dark respiration which was associated with nitrogen fixation in Synechococcus RF-1. Twenty-five millimolar NaNO3 added 30 min before the start of dark period suppressed this respiratory increase. If 1.25 mM CaCl2 was added to the EGTA-treated sample from 3 to at least 10 h later in the dark period, a quick rise in respiratory rate was observed. This rise was also reduced by 25 mM NaNO3. Extracellular Ca2+ appears to be required for the increase in dark respiration associated with the rhythmic appearance of nitrogenase activity in the dark cycle.