The systematic evaluation of different transthoracic echocardiographic (TTE) methods to determine cardiac output (CO) and the effect of changes in intravascular volume on echocardiographically determined indexes of cardiovascular structure in the rat has not been documented. With the use of 11 Wistar rats, simultaneous echocardiographic and thermodilution measurements of CO were compared at baseline and after blood withdrawal or transfusion at 43 different levels of intravascular volume and using 10 different echocardiographic approaches. The best correlation (r = 0.93; P < 0.0001), least bias (-3 ml/min), and best precision (16 ml/min) between thermodilution and echocardiographic methods were obtained at the level of aortic annulus using pulsed Doppler. In conclusion, CO could be accurately assessed in rats using TTE and pulsed Doppler at the level of the aortic annulus. This annulus was demonstrated to remain stable, but pulmonary annulus, thoracic aorta, mitral valve, and left ventricular diameters were found to be more modifiable during volumic changes.