The release of amino acids from their vacuolar store was studied in situ, i.e. in cells with selectively permeabilized plasma membrane and functionally intact vacuoles. As we previously described [Roos et al., J. Biol. Chem. 272 (1997) 15849-15855], this transport process is regulated by extravacuolar adenylates at their physiological concentrations. We now show, using our test object Penicillium cyclopium, that not only purine but also pyrimidine nucleotides are involved in the control of efflux of vacuolar phenylalanine. At 0.1 mM adenosine or guanosine phosphates inhibit, whereas cytidine or uridine phosphates stimulate the rate of efflux. At 1 mM the same nucleotides have no measurable impact on efflux but abolish the effects of other nucleotides present at 0.1 mM. This argues for at least two interacting binding sites with different nucleotide affinities. The minimum structural requirement for any of the observed effects is a non-cyclic ribonucleoside monophosphate. In intact cells, cytosolic concentrations of ATP (representing purine nucleotides) and CTP (representing pyrimidine nucleotides) are 1-2 mM and 0.05-0.2 mM, respectively. ATP is therefore assumed to dominate transport control and allow optimum efflux (and uptake) rates. Short-time starvation of carbon and nitrogen adjusts CTP and ATP at levels that cause declining efflux rates. During prolonged starvation both nucleotides fall below their transport-controlling concentrations and thus allow increasing rates of efflux from the still maintained vacuolar pool. Hence, efflux control under nutrient limitation includes an interplay of purine and pyrimidine nucleotides which precisely regulates the release of vacuolar amino acids and enables flexible adjustment to either amino acid saving or cell survival.