Titin is a giant protein responsible for passive-tension generation in muscle sarcomeres. Here, we used single-molecule AFM force spectroscopy to investigate the mechanical characteristics of a recombinant construct from the human cardiac-specific N2B-region, which harbors a 572-residue unique sequence flanked by two immunoglobulin (Ig) domains on either side. Force-extension curves of the N2B-construct revealed mean unfolding forces for the Ig-domains similar to those of a recombinant fragment from the distal Ig-region in titin (I91-98). The mean contour length of the N2B-unique sequence was 120 nm, but there was a bimodal distribution centered at approximately 95 nm (major peak) and 180 nm (minor peak). These values are lower than expected if the N2B-unique sequence were a permanently unfolded entropic spring, but are consistent with the approximately 100 nm maximum extension of that segment measured in isolated stretched cardiomyofibrils. A contour-length below 200 nm would be reasonable, however, if the N2B-unique sequence were stabilized by a disulphide bridge, as suggested by several disulphide connectivity prediction algorithms. Since the N2B-unique sequence can be phosphorylated by protein kinase A (PKA), which lowers titin-based stiffness, we studied whether addition of PKA (+ATP) affects the mechanical properties of the N2B-construct, but found no changes. The softening effect of PKA on N2B-titin may require specific conditions/factors present inside the cardiomyocytes.