The purpose of this study was to define the validation methods and outcomes of a conductance catheter system specifically for in vivo murine cardiac hemodynamic analysis. To express the relationship between conductance and blood volumes, we used an in vitro model to derive a volume-conductance line. The volume-conductance line was used to compute raw volume from the modified conductance signals. The parallel volume was calibrated with hypertonic (15%) saline injected from extrajugular vein. The ventricular volume was computed by raw volume minus parallel volume. The accuracy of conductance volumetric measurements was validated with a static in situ infusion of calibrated volumes of whole blood injected into arrested left ventricles. In vivo dynamic measurements were performed with 24 C57B1/6 mice, 6 months old; for comparison of established values. The in situ model showed that after calibration, the experimental coefficient, alpha, was equal to 1 and the measured volume by conductance catheter was equal to the true volume of the left ventricle (y = 0.982x + 0.513, p < .0001). For the in vivo models, the end-diastolic volumes and the stroke volumes and cardiac output determined with the conductance catheter system were 17.3 +/- 1.0 microL, 10.6 +/- 0.9 microL, and 6.0 +/- 0.5 mL/min, respectively. We validated the relationship between measured volume by conductance catheter and the true volume and demonstrated the accuracy of the volume-conductance line for conversion of conductance to volume.