Strain determination in fetal hearts is essential but conventional methods do not provide opportunities for orthogonal strain analysis and require the use of EKG-gating. A new non-gated 4D echocardiography method was tested for accuracy in strain computation. Fifteen rabbit hearts were studied. Each heart was mounted in a water tank to facilitate ultrasound scanning, connected to a calibrated pump by a balloon sutured into the left ventricle (LV), and pumped at Stroke Volumes (SV) 1-5 ml and Stroke Rates (SR) 40 and 80 bpm. Three 0.7mm sonomicrometry crystals were secured in the myocardium to conduct longitudinal strain (LS) and circumferential strain (CS) measurements. At each SV and each SR, 4D images were obtained by an X6-1 probe interfaced with the Philips iU-22 ultrasound system while sonomicrometry displacement was recorded. This process was performed pre and post simulated myocardial infarction (MI). 4D images were analyzed offline for strain by a MATLAB-based program. 4D echocardiography- derived strain data correlated with sonomicrometry-derived strain at each SV (CS: R2; = 0.91, p<0.05; LS: R2; = 0.87, p<0.05). A decrease in strain post-MI was detected by both echocardiography and sonomicrometry. Non-gated 4D echocardiography is an accurate method for strain determination of fetal hearts.