Pichia sorbitophila is a halotolerant yeast capable of surviving to extracellular NaCl concentrations up to 4 M in mineral medium when glucose or glycerol are the only carbon and energy sources. Evidence is presented here that glycerol, the main compatible solute this yeast accumulates so as to maintain osmotic balance, is actively co-transported with protons. This transport system was shown to be constitutive, not needing induction by either glycerol or salt, and was not repressible by glucose. In glucose- or glycerol-grown cells, a simple diffusion was detectable, and iterative calculations were performed to calculate kinetic parameters, in the presence and in the absence of NaCl. At 25 degrees C, pH 5.0, in glucose-grown cells these were: Km = 0.81 +/- 0.11 mM and Vmax = 634.2 +/- 164.8 mumol h-1 per g (glycerol); Km = 1.28 +/- 0.60 mM and Vmax = 558.6 +/- 100.6 mumol h-1 per g (protons). Correspondent stoichiometry was approximately 1, either for these conditions or in the presence of 1 M-NaCl. An increase in accumulation capacity was evident when different concentrations of NaCl were present. This capacity was shown to be dependent on delta pH and membrane potential, consistently with an electrogenic character. We suggest that the main role of this system is in osmoregulation, by keeping glycerol accumulated inside the cells, compensating for leakage, due to its liposoluble character.