Transcriptional analysis of the region downstream of the pcbAB gene (which encodes the alpha-aminoadipyl-cysteinyl-valine synthetase involved in cephalosporin synthesis) of Acremonium chrysogenum revealed the presence of two different transcripts corresponding to two new ORFs. ORF3 encodes a putative D-hydroxyacid dehydrogenase and cefT (for transmembrane protein) encodes a multidrug efflux pump belonging to the Major Facilitator Superfamily (MFS) of membrane proteins. The CefT protein has 12 transmembrane segments (TMS) and contains motifs A, B, C, D2 and G characteristic of the Drug:H(+) antiporter 12-TMS group of the major facilitator superfamily. The CefT protein confers resistance to some toxic organic acids, including isovaleric acid and phenylacetic acid. Targeted inactivation of ORF3 and cefT by gene replacement showed that they are not essential for cephalosporin biosynthesis. However, amplification of the cefT gene results in increments of up to 100% in cephalosporin production in the A. chrysogenum C10 strain. Amplification of a truncated form of the cefT insert did not lead to cephalosporin overproduction. It seems that the CefT protein is involved in cephalosporin export from A. chrysogenum or in transmembrane signal transduction, and that there are redundant systems involved in cephalosporin export.