Ankyrins are a ubiquitously expressed family of intracellular adaptor proteins involved in targeting diverse proteins to specialized membrane domains in both the plasma membrane and the endoplasmic reticulum. We described here that the C-terminal domain of ankyrin-B interact specifically with Z-line portion of titin in yeast two-hybrid analysis, in vitro pull-down assays and localization experiments in COS-7 cells. In this study we provide the first experimental evidence that Z-line portion of titin is necessary for the localization of ankyrin-B and ankyrin-B links between the sarcolemma and the myofibril in costameres.