Cytosolic free calcium ([Ca2+]c) has been measured in the mycelial fungus Neurospora crassa with Ca2(+)-selective microelectrodes. The mean value of [Ca2+]c is 92 +/- 15 nM and it is insensitive to external pH values between 5.8 and 8.4. Simultaneous measurement of membrane potential enables the electrochemical potential difference for Ca2+ across the plasma membrane to be estimated as about -60 kJ.mol-1-a value that cannot be sustained either by a simple Ca2(+)-ATPase, or, in alkaline conditions, by straightforward H+/Ca2+ exchange with a stoichiometric ratio of less than 5 H+/Ca2+. We propose that the most likely alternative mechanism of Ca2+ efflux is ATP-driven H+/Ca2+. In accord with this proposal, depletion of the ATP level from 2.5 to 0.5 mM by CN- elicits an increase in [Ca2+]c, but only in alkaline conditions in which the putative H+/Ca2(+)-ATPase would be selectively stalled. The insensitivity of Ca2+ homeostasis to CN- in more acid conditions implies that the Km (ATP) of the transport system is 100 microM or less. The increase in [Ca2+]c in the presence of CN- at pH 8.4 (50 nM.min-1) is compared with 45Ca2+ influx (0.62 mM.min-1) under the same conditions. The proportion of entering Ca2+ remaining free in the cytosol is only 8 x 10(-5), and since the concentration of available chelation sites on Ca2(+)-binding proteins is unlikely to exceed 100 microM, a major role for the fungal vacuole in short-term Ca2+ homeostasis is indicated. This notion is supported by the observation that cytosolic Ca2+ homeostasis is disrupted by a protonophore, which rapidly abolishes the driving force (a transmembrane pH difference) for Ca2+ uptake into fungal vacuoles.