Sec20p is a component of the yeast Saccharomyces cerevisiae secretory pathway that does not have a close homolog in higher eukaryotic cells. To verify the function of Sec20p in other fungal species, we characterized the gene encoding a Sec20p homolog in the human fungal pathogen Candida albicans. The deduced protein has 27% identity with, but is missing about 100 N-terminal residues compared to S. cerevisiae Sec20p, which is part of the cytoplasmic tail interacting with the cytoplasmic protein Tip20p. Because a strain lacking both C. albicans SEC20 alleles could not be constructed, we placed SEC20 under transcriptional control of two regulatable promoters, MET3p and PCK1p. Repression of SEC20 expression in these strains prevented (MET3p-SEC20 allele) or retarded (PCK1p-SEC20 allele) growth and led to the appearance of extensive intracellular membranes, which frequently formed stacks. Reduced SEC20 expression in the PCK1p-SEC20 strain did not affect morphogenesis but led to a series of hypersensitivity phenotypes including supersensitivity to aminoglycoside antibiotics, to nystatin, to sodium dodecyl sulfate, and to cell wall inhibitors. These results demonstrate the occurrence and function of Sec20p in a fungal species other than S. cerevisiae, but the lack of the N-terminal domain and the apparent absence of a close TIP20 homolog in the C. albicans genome also indicate a considerable diversity in mechanisms of retrograde vesicle traffic in eukaryotes.