In the prokaryote Synechococcus RF-1, circadian changes in the uptake of L-leucine and 2-amino isobutyric acid were observed. Uptake rates in the light period were higher than in the dark period for cultures entrained by 12/12 hour light/dark cycles. The periodic changes in L-leucine uptake persisted for at least 72 hours into continuous light (L/L). The rhythm had a freerunning period of about 24 hours in L/L at 290C. A single dark treatment of 12 hours could initiate rhythmic leucine uptake in an L/L culture. The phase of rhythm could be shifted by a pulse of low temperature (0°C). The free-running periodicity was "temperature-compensated" from 21 to 370C. A 24 hour depletion of extracellular Ca2+ before the free-running L/L condition reduced the variation in uptake rate but had little effect on the periodicity of the rhythm. The periodicity was also not affected by the introduction of 25 mm NaNO3. The uptake rates for 20 natural amino acids were studied at 12 hour intervals in cultures exposed to 12/12 hour light/dark cycles. For eight of these amino acids Circadian rhythms have been documented for organisms throughout the eukaryotic kingdoms (3). Circadian rhythmicity had not been detected in any prokaryote until recently, when endogenous circadian timing in nitrogen-fixation (5,8,12) and cell division (12,16) were reported in some strains of unicellular cyanobacteria belonging to the genus Svnechococcus.In eukaryotic organisms, endogenous circadian changes in the uptake rates of histidine and lysine have been observed in the yeast Saccharomyces cerevisiae (4).In this report, we investigated the uptake rates of natural amino acids in the prokaryotic Synechococcus RF-1 in an attempt to observe endogenous circadian variations. For comparison, we also investigated uptake rates of L-leucine in Svnechococcuis PCC 7942 and Synechocystis PCC 6803. MATERIALS AND METHODSSvnechococcus RF-1 (PCC 8801) was cultured as described previously (8 When cells of Synechococcus RF-1 were cultured under 12/ 12 h L/D cycles, the uptake rates of leucine (Fig. 1A) and non-metabolizable AIB (Fig. 2) fluctuated periodically and were several times higher during the light period than during the dark period. After the cultures were subsequently exposed to L/L, the periodic variations in leucine and AIB uptake persisted for at least 72 h (Fig. 1B) and 60 h ( Fig. 2A), respectively, without a noticeable change in periodicity. The average rhythm period under free-running conditions was about 24 h. The amino acid uptake rhythm was about 12 h out of phase with the nitrogen-fixation (5,8)