Objectives: Indirect evidence suggests a role for Ca²⁺-overload in electrical and mechanical alterations caused by atrial tachycardia. The present study assessed the alterations in cellular [Ca²⁺] and contractile function caused by rapid atrial cellular activation. Methods: Intracellular Ca²⁺ transients (CaT) and cell shortening (CS) were measured by microfluorometry (Indo-1 AM) and video edge-detection in isolated, field-stimulated canine atrial myocytes (37°C). Results: Abrupt increases in frequency (0.3–3 Hz) caused rapid increases in diastolic [Ca²⁺]_i (DCa) that were maintained during rapid-pacing for up to 50 min. When short-term (3-min) rapid-pacing was imposed, CaT and CS increased initially upon returning to 0.3 Hz, but then declined rapidly to 64±5 and 49±7%, respectively, of pre-tachycardia values, returning to control after ∼15 min. Post-tachycardia CaT and CS reductions were prevented by decreasing [Ca²⁺]_o during tachycardia to prevent Ca²⁺-overload. CS reductions correlated with indices of Ca²⁺ loading during tachycardia. Restoration of CaT to normal during post-tachycardia contractile dysfunction (by increasing [Ca²⁺]_o) returned CS to normal, indicating that reduced Ca²⁺ release, not reduced myofilament Ca²⁺-sensitivity, caused post-tachycardia contractile failure. Estimation of sarcoplasmic-reticulum Ca²⁺-stores (caffeine-induced Ca²⁺-release) confirmed tachycardia-induced Ca²⁺-loading and suggested that reduced Ca²⁺-stores decreased Ca²⁺-release post-tachycardia. Conclusions: Atrial tachycardia increases cellular Ca²⁺-loading, leading to post-tachycardia abnormalities in Ca²⁺-handling that produce contractile dysfunction. These findings are the first direct evidence for the frequently-postulated role of Ca²⁺-overload in tachycardia-induced abnormalities of atrial function.