Saccharomyces cerevisiae Nfs1p is mainly found in the mitochondrial matrix and has been shown to participate in iron-sulfur cluster assembly. We show here that Nfs1p contains a potential nuclear localization signal, RRRPR, in its mature part. When this sequence was mutated to RRGSR, the mutant protein could not restore cell growth under chromosomal NFS1-depleted conditions. However, this mutation did not affect the function of Nfs1p in biogenesis of mitochondrial iron-sulfur proteins. The growth defect of the mutant was complemented by simultaneous expression of the mature Nfs1p, which contains the intact nuclear localization signal but lacks its mitochondrial-targeting presequence. These results suggest that a fraction of Nfs1p is localized in the nucleus and is essential for cell viability.