Glycosylphosphatidylinositols (GPIs) are essential for viability in yeast and have key roles in cell wall construction. Assembly of Saccharomyces cerevisiae GPIs includes the addition of a fourth, side-branching mannose (Man) to the third Man of the core GPI glycan by the Smp3 mannosyltransferase. The SMP3 gene from the human pathogenic fungus Candida albicans has been cloned. CaSMP3 complements the inviable S. cerevisiae smp3 null mutant and, when expressed in an S. cerevisiae smp3/gpi13 double mutant, it permits in vivo conversion of the Man3-GPI precursor that accumulates in that mutant to a Man4-GPI. One allele of CaSMP3 was disrupted using the ura-blaster procedure, then the remaining allele was placed under the control of the glucose-repressible MAL2 promoter. Repression of CaSMP3 expression leads to accumulation of a GPI precursor glycolipid whose glycan headgroup contains three mannoses and bears a phosphodiester-linked substituent on its first Man. Under repressing conditions, cells exhibited morphological and cell wall defects and became inviable. CaSmp3p therefore adds a fourth, alpha1,2-linked Man to trimannosyl GPI precursors in C. albicans and is necessary for viability. Because addition of a fourth Man to GPIs is of less relative importance in mammals, Smp3p is a potential antifungal target.