Candida albicans is the most prevalent human pathogenic fungus and causes superficial, mucosal or systemic infections in immunocompromised patients. The recent sequencing of the C. albicans genome and the development of new molecular methodologies have been allowing the analysis and identification of new virulence factors. We have developed a transposon for production of random fusions of a sequence coding for GFP (green fluorescent protein) in the genome or in a specific cloned gene. The transposon mTn5-CaGFP contains in its ends the bacterial Tn5 inverted repeats, the entire coding region for a C. albicans codon-optimized GFP without the start codon. The transposon also contains a kanamycin resistance and URA3 genes for selection in E. coli and C. albicans, respectively. The transposition reaction was done in-vitro using the Tn5 transposase, a transposon PCR product as a donor, and a plasmid bearing the essential CDC3 septin gene as a target. After bacterial transformation, plasmid DNA was purified from the selected clones. By restriction analysis we found that, of 96 clones obtained, 40 carried transposon insertions in CDC3 and the DNA sequencing revealed that 6 clones had in-frame GFP fusions. These 6 insertion alleles were transferred to C. albicans diploid genome by homologous recombination after digestion with the NotI to release the insert. The subcellular localization of the septin-GFP fusion proteins was evaluated by fluorescence microscopy after growing the cells in three different growth conditions: 30ºC (yeast) or 37ºC with (hyphae induction) or without serum (pseudohyphae induction). Two individual insertions near the C-terminal region gave rise to cells with wild type morphologies and with septin-GFP fusion protein localized to the typical ring at the bud neck. But in some cells we also detected irregular septin-GFP localization in patches at the growing bud tip. In contrast, the other 4 insertions in the central domain or in the N-terminal region gave rise to dominant phenotypes: elongated buds in cells growing at 30C and incomplete citokinesis in pseudohyphal cells. We were not able to detect GFP signals in these mutants, indicating that either the fusion proteins are not targeted to their proper localization or they are unstable. Thus, by generating a set of individual GFP fusions throughout the protein sequence, this transposon may serve as a versatile tool to dissect protein domain function and protein subcellular localization. The addition of a recycling marker into the transposon will allow the mutation of the second allele of the target gene.