Abstract: Coenzyme A (CoA) is a ubiquitous cofactor required for numerous enzymatic carbon group transfer reactions. CoA biosynthesis requires contributions from various amino acids with pantothenate as an important intermediate which can be imported from the medium or synthesized de novo. Investigating function and expression of structural genes involved in CoA biosynthesis of the yeast Saccharomyces cerevisiae, we show that deletion of ECM31 and PAN6 results in mutants requiring pantothenate while loss of PAN5 (related to panE from E. coli) still allows prototrophic growth. A temperature-sensitive mutant defective for fatty acid synthase activity could be functionally complemented by a gene significantly similar to eukaryotic pantothenate kinases (YDR531W). Enzymatic studies and heterologous complementation of this mutation by bacterial and mammalian genes showed that YDR531W encodes a genuine pantothenate kinase (new gene designation: CAB1, "coenzyme A biosynthesis"). A G351S missense mutation within CAB1 was identified to cause the conditional phenotype of the mutant initially studied. Similar to CAB1, genes YIL083C, YKL088W, YGR277C and YDR106C responsible for late CoA biosynthesis turned out as essential. Null mutants could be complemented by their bacterial counterparts coaBC, coaD and coaE, respectively. Comparative expression analyses showed that some CoA biosynthetic genes are weakly de-repressed with ethanol as a carbon source compared with glucose.