Zygosaccharomyces bailii ISA 1307 displays biphasic growth in a medium containing a mixture of glucose (0.5%, w/v) and acetic acid (0.5%, w/v), pH 5.0 and 3.0. In cells harvested during the first growth phase, no activity of a mediated acetic acid transport system was found. Incubation of these cells in phosphate buffer with cycloheximide for 1 h restored activity of an acetic acid carrier which behaved as the one present in glucose-grown cells. These results indicated that the acetic acid carrier is probably present in cells from the first growth phase of the mixed medium but its activity was affected by the presence of acetic acid in the culture medium. In glucose-grown cells, after incubation in phosphate buffer with glucose and acetic acid, the activity of the acetic acid carrier decreased significantly with increased acid concentration in the incubation buffer. At acid concentrations above 16.7 mM, no significant carrier activity was detectable. Furthermore, the intracellular acid concentration increased with the extracellular one and was inversely correlated with the activity of the acetic acid carrier, suggesting the involvement of a feedback inhibition mechanism in the regulation of the carrier. During biphasic growth, the first phase corresponded to a simultaneous consumption of glucose and acetic acid, and the second to the utilization of the remaining acid. The enzyme acetyl-CoA synthetase was active in both growth phases, even in the presence of glucose. Activity of isocitrate lyase and phosphoenolpyruvate carboxykinase was found only in acetic-acid-grown cells. Thus it appears that both membrane transport and acetyl-CoA synthetase and their regulation are important for Z. bailii to metabolize acetic acid in the presence of glucose. This fact correlates with the high resistance of this yeast to environments with mixtures of sugars and acetic acid such as those often present during wine fermentation.