Lysine biosynthesis in yeast requires the posttranslational conversion of the alpha-aminoadipate semialdehyde reductase Lys2 by the 4'-phosphopantetheinyl transferase (PPTase) Lys5 from the inactive apo-form into the catalytically active holo-form. In this reaction, the peptidyl carrier domain of Lys2 is modified at a conserved serine residue side chain with the 4'-phosphopantetheine moiety derived from coenzyme A. We have deleted the lys5 gene in Saccharomyces cerevisiae to investigate the substrate specificity of various heterologous PPTase genes of bacterial and fungal origin by testing their ability to complement lys5 in trans. Genes encoding PPTases Sfp and Gsp from Bacillus spp., which are involved in non-ribosomal peptide antibiotic synthesis, complemented the lys5 deletion, whereas ydcB of Bacillus subtilis, which encodes the acyl carrier protein synthase involved in fatty acid synthesis, could not. Two yet uncharacterized fungal genes, q10474 of Schizosaccharomyces pombe, meanwhile annotated as the putative lys7 gene, and npgA of Aspergillus nidulans, also complemented the lys5 deletion and have thus been functionally characterized as PPTases. The complementation system described also provides the basis for a simple method of functional characterization of PPTase candidate genes and their cloning from chromosomal DNA or cDNA libraries of diverse origin.