In the yeast Saccharomyces cerevisiae, the paralogous genes ALT1 and ALT2 have been proposed to encode alanine aminotransferase isozymes. Although in other microorganisms this enzyme constitutes the main pathway for alanine biosynthesis, its role in S. cerevisiae had remained unclear. Results presented in this paper show that under respiratory conditions, Alt1p constitutes the sole pathway for alanine biosynthesis and catabolism, constituting the first example of an alanine aminotransferase that simultaneously carries out both functions. Conversely, under fermentative conditions, it plays a catabolic role and alanine is mainly synthesized through an alternative pathway. It can thus be concluded that ALT1 has functions in alanine biosynthesis and utilization or only alanine utilization under respiratory and fermentative conditions, respectively. ALT2 expression was repressed under all tested conditions, suggesting that Alt2p biosynthesis is strictly controlled and only allowed to express under peculiar physiological conditions.