The effect of protamine sulfate on left ventricular function was evaluated in 23 patients undergoing heart surgery requiring cardiopulmonary bypass (CPB). Ventricular function was assessed by measuring cardiac index with a thermodilution pulmonary artery catheter and fractional shortening with transesophageal echocardiography (TEE). After CPB, a heparin-neutralizing dose of protamine was infused continuously for 5 min. Hemodynamic variables were obtained and fractional shortening was measured before protamine administration (time 0), during protamine infusion (2.5 and 5 min), and after the infusion (10 and 15 min after the start of protamine). Patients with a preoperative ejection fraction ≥ 50% had no statistically significant changes in either cardiac index or fractional shortening with protamine administration, when compared to time 0. However, patients with a preoperative ejection fraction < 50% had a significant decrease in fractional shortening which occurred during and immediately after protamine administration when compared to time 0 (P < 0.01), with no associated change in hemodynamics. Our results suggest that protamine may have a negative inotropic effect that is apparent in patients with impaired ventricular function.