Appropriate nuclear membrane structure is important for all eukaryotic organisms as evidenced by the numerous human diseases and alterations in gene expression caused by inappropriate targeting of proteins to the inner nuclear membrane (INM). We report here the first genome-wide screen to identify proteins functioning in INM targeting. We transformed to near completion the 4850 members of the Saccharomyces cerevisiae deletion collection of unessential genes in the 96-well format with a plasmid encoding a reporter protein, Trm1-II-GFP, which normally resides at the INM. We found that deletion of genes encoding subunits of the N-terminal acetyltransferase, NatC, cause mislocation of Trm1-II-GFP from the INM to the nucleoplasm. Mass spectroscopic analysis indicates that Trm1-II-GFP is N-acetylated. N-terminal mutations of Trm1-II-GFP predicted to ablate N-acetylation cause nucleoplasmic location, whereas a variant with an N-terminal alteration predicted to allow N-acetylation by NatC is located at the INM, providing genetic support that Trm1p-II N-acetylation is necessary for its subnuclear INM location. However, because N-acetylation appears not to be sufficient for INM targeting, it may provide a necessary role for INM targeting by affecting Trm1-II-GFP structure and exposure of cis-acting INM targeting motifs. We also discovered that YIL090W/Ice2p, an integral membrane protein located in the endoplasmic reticulum, is necessary for efficient targeting of Trm1-II-GFP to the INM. YIL090W/Ice2p may serve as a tether for INM proteins or as a regulator of INM tethers. Our methodology can be extrapolated to obtain genome-wide perspectives of mechanisms necessary to achieve appropriate subcellular and/or suborganellar location for any resident protein.