Carnitine is an essential metabolite that enables intracellular transport of fatty acids and acetyl units. Here we show that the yeast Candida albicans can synthesize carnitine de novo, and we identify the 4 genes of the pathway. Null mutants of orf19.4316 (trimethyllysine dioxygenase), orf19.6306 (trimethylaminobutyraldehyde dehydrogenase), and orf19.7131 (butyrobetaine dioxygenase) lacked their respective enzymatic activities and were unable to utilize fatty acids, acetate, or ethanol as a sole carbon source, in accordance with the strict requirement for carnitine-mediated transport under these growth conditions. The second enzyme of carnitine biosynthesis, hydroxytrimethyllysine aldolase, is encoded by orf19.6305, a member of the threonine aldolase (TA) family in C. albicans. A strain lacking orf19.6305 showed strongly reduced growth on fatty acids and was unable to utilize either acetate or ethanol, but TA activity was unaffected. Growth of the null mutants on nonfermentable carbon sources is restored only by carnitine biosynthesis intermediates after the predicted enzymatic block in the pathway, which provides independent evidence for a specific defect in carnitine biosynthesis for each of the mutants. In conclusion, we have genetically characterized a complete carnitine biosynthesis pathway in C. albicans and show that a TA family member is mainly involved in the aldolytic cleavage of hydroxytrimethyllysine in vivo.—Strijbis, K., van Roermund, C. W. T., Hardy, G. P., van den Burg, J., Bloem, K., de Haan, J., van Vlies, N., Wanders, R. J. A., Vaz, F. M., Distel, B. Identification and characterization of a complete carnitine biosynthesis pathway in Candida albicans.