Lamin A and some integral membrane proteins of the nuclear envelope (NE) have been linked to human diseases, mostly dystrophies. To comprehensively identify integral membrane proteins specific to the nuclear envelope, we have carried out a subtractive proteomics analysis of NEs isolated from rodent liver using Multidimensional Protein Identification Technology (MudPIT). An NE fraction and a nucleus-depleted membrane fraction were separately analyzed by MudPIT and proteins appearing in both fractions were 'subtracted' from the NE fraction. This identified 67 novel putative NE transmembrane proteins in addition to the 13 that had been previously characterized. Most or all of the new proteins we identified are likely to be bona fide NE Transmembrane proteins (NETs), since all eight of the first group of proteins we tested in a cell transfection assay target to the NE. Moreover, five of the eight NETs remained associated with the nuclear periphery after extraction with Triton-X100, suggesting an association with the nuclear lamin polymer. 27 of the proteins occur in chromosomal regions where 18 different human dystrophies have been mapped, making these proteins disease candidates. We have analysed the expression of these proteins using transcriptome databases, providing direction for future functional analysis of these novel proteins.