β-Adrenergic stimulation of cardiac muscle activates protein kinase A (PKA), which is known to phosphorylate proteins on the thin and thick filaments of the sarcomere. Cardiac muscle sarcomeres contain a third filament system composed of titin, and here we demonstrate that titin is also phosphorylated by the β-adrenergic pathway. Titin phosphorylation was observed after β-receptor stimulation of intact cardiac myocytes and incubation of skinned cardiac myocytes with PKA. Mechanical experiments with isolated myocytes revealed that PKA significantly reduces passive tension. In vitro phosphorylation of recombinant titin fragments and immunoelectron microscopy suggest that PKA targets a subdomain of the elastic segment of titin, referred to as the N2B spring element. The N2B spring element is expressed only in cardiac titins, in which it plays an important role in determining the level of passive tension. Because titin-based passive tension is a determinant of diastolic function, these results suggest that titin phosphorylation may modulate cardiac function in vivo.