Tissue transglutaminase (TGM2; also known as TG2 or tTG) localizes to the syncytial microvillous membrane (MVM) of the human placenta, the primary interface between maternal and fetal tissue. To identify TGM2 substrates in the MVM, membrane vesicles were prepared and labeled with biotinylated acyl donor or acceptor probes. Biotinylated species were selected on an avidin affinity matrix and identified by mass spectrometry of tryptic peptides. The most abundant were cytoskeletal (actin, tubulin, and cytokeratin) and membrane-associated (annexins, integrins, and placental alkaline phosphatase) proteins. During pregnancy, apoptotic particulate material, the end product of the trophoblast life cycle, is shed from the MVM into maternal circulation. Shed material was isolated from primary trophoblast cultures in which syncytial-like masses develop by fusion. A substantial fraction of actin in the particles was in the form of covalent polymeric aggregates, in contrast to cellular actin, which dissociated completely into monomer in SDS-PAGE. When cells were cultured in the presence of transglutaminase inhibitors, actin in the shed particles remained exclusively in monomeric form, and a reduction in trophoblast intercellular fusion and differentiation was observed. These findings suggest that transglutaminase-mediated cross-linking stabilizes the particulate material shed from the placenta.