The analysis of clones obtained by rapid amplification of the 5' end and by primer extension of the mRNA for carrot bifunctional dihydrofolate reductase-thymidylate synthase showed transcripts of differing lengths that belonged to two sub-populations. The longer transcripts were found to contain a translation start site 147 nt upstream of, and in frame with, the one which is present in the shorter transcripts. The ORF that begins at this ATG codes for a protein of 64714 Da, which is much larger than mature DHFR-TS subunit. The N-terminus region of this polypeptide shows features typical of plant transit peptides. Immunogold labelling studies and immunorecognition of the plastid-containing sub-cellular fraction suggested a plastidial localisation of the bifunctional protein. Although plant cells were shown to contain folate pools in plastids, in mitochondria and in the cytosol, few enzymes of the folate pathway have been associated with any sub-cellular compartment. Thus, this is the first indication for the presence of an enzyme of the folate biosynthetic pathway in plastids. The longer transcripts revealed the presence of a TC microsatellite at the 5'-untranslated end.