The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier forming proteins like occludin and the claudin family, scaffolding proteins like ZO-1 and some cytoskeletal, signaling and cell polarity proteins. To define a more complete list of proteins and investigate their functional implications, we identified the proteins which are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N- or C-terminus, expressing these fusion proteins in MDCK epithelial cells and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Out of a predicted proteome of ≈ 9000 we identified more than 300 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C- terminal ends. Those proximal to the N-terminus were enriched in transmembrane tight junction proteins and those proximal to the C-terminus were enriched in cytoskeletal proteins. We also identified many unexpected, but easily rationalized proteins. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. This approach is being extended to identify proteins which are near occludin and claudins with the goal of defining subjunctional protein compartments.