Abstract: Hyphal morphogenesis in Candida albicans is regulated by multiple pathways, which act either by inducing or repressing filamentation. Most notably Tup1, Nrg1, and Rfg1 are transcriptional repressors while Efg1, Flo8, Cph1, and Czf1 can induce filamentation. Here we present the functional analysis of CaSFL1, which encodes the C. albicans homolog of the Saccharomyces cerevisiae SFL1 (suppressor of flocculation) gene. Deletion of CaSFL1 results in flocculation (i.e. the formation of clumps) of yeast cells, which is most pronounced in minimal medium. The flocs contained hyphae already under non-inducing conditions and filamentation could be enhanced with hyphal inducing cues at 37 degrees C. Expression of SFL1 in a heterozygous mutant under the control of the CaMET3 promoter was shown to complement these defects and allowed switching between wild type and mutant phenotypes. Interestingly, increased expression of SFL1 using a MET3prom-SFL1 construct prior to the induction of filamentation completely blocked germ tube formation. To localize Sfl1 in vivo we generated a SFL1-GFP fusion. Sfl1-GFP was found in the nucleus in both yeast and to a lesser extent in hyphal cells. Using RT-PCR we find an increased expression of ALS1, ALS3, HWP1, ECE1, and also FLO8. Our results suggest that Sfl1 functions in the repression of flocculation and filamentation and thus represents a novel negative regulator of C. albicans morphogenesis.