The sarcomeric Z-disk contains various proteins thought to be involved in mechanical signaling, but little is known about the properties of a distinct region at the Z-disk periphery, the N1-line. At this region, the inextensible Z-disk-bound portion of titin adjoins the extensible I-band titin segment. Titin at the Z/I junction comprises four Ig-domains (Z7-I1) interspersed with unique sequences, encoded by titin-exons 27/28. We hypothesize that the N1-line titin region participates in mechanical signaling via protein-protein interactions and post-translational modifications. Yeast-two-hybrid screens "fished" various potential interactors of N1-line titin out from a human cardiac cDNA library, including signaling and structural proteins such as atrial natriuretic peptide, myospryn, and tubulin-8. Additionally, N1-line titin interacted in yeast with the I-band titin Ig-domains I13 and I14, suggesting this part of the elastic titin could bend back to the Z/I-junction to bind N1-line titin; this could be accomplished by the truncated novex-3 titin isoform. Furthermore, we identified two serines within N1-line titin, S2078 and S2080, which were phosphorylated in a large majority of ~30 adult mouse hearts studied by mass spectrometry. In-vitro phosphorylation tests demonstrated that protein kinases, cdc2 and ERK2 (but not PKA or CaMKII), can phosphorylate S2078. We speculate that phosphorylation of N1-line titin might be important for reversible protein-protein interactions, like those required in mechanical signaling events. N1-line titin phosphorylation could also be necessary for the development of the Z/I-brush during myofibrillogenesis. We conclude that titin's N1-line region may be part of a dynamically regulated protein network important for myocyte mechanical function.