The I-band portion of the giant muscle protein titin is elastic but detailed knowledge of titin arrangement and interactions in this region is lacking. We determined whether or not select domains from this region can associate with one another, as observed for titin's "distal" immunoglobulin domain-like (Ig-)region. Using sedimentation-velocity centrifugation and size-exclusion chromatography of purified human titin fragments, we detected a monomeric state of the titin N2B-domain or the N2B-unique sequence contained therein. The constitutively expressed PEVK-domain and the Ig-only fragment I9-I12 also behaved as monomers in-vitro, whereas the N2A-segment showed both dimeric and monomeric behavior. Yeast-2-hybrid and GST-pulldown interaction tests demonstrated that both the N2B and the N2A domain, but not the PEVK-fragment, bind to the small heat-shock proteins (sHSPs), alphaB-crystallin and HSP27. These interactions were confirmed on isolated human cardiac or rabbit psoas myofibrils incubated ex-vivo with either sHSP type. Stretching myofibrils to promote titin-domain unfolding increased the propensity of sHSPs to bind I-band titin. Protein unfolding in-vitro by 8 M urea caused aggregation of the N2A-segment under acidic conditions (pH 6.7), but not at pH 7.2. Importantly, alphaB-crystallin prevented the aggregation of the N2A-segment at pH 6.7 partially (molar ratio of N2A:alphaB-crystallin, 1:5) or fully (ratio, 1:10); HSP27 did not have this protective effect. In cultured neonatal rat cardiomyocytes, the two sHSPs translocated from the cytosol to the sarcomeric Z-disk/I-band region on inhibition of the proteasome. In adult rat cardiomyocytes both sHSPs associated with I-band titin already under normal culture conditions, suggesting an age-related increase in chaperoning activity. We conclude that (1) titin filaments may run through the elastic segment mainly as monomers; (2) sHSPs associate with elastic titin domains under various stress conditions; and (3) sHSPs like alphaB-crystallin are able to protect I-band titin regions from aggregation under adverse intracellular circumstances.