To compare the image quality, radiation dose and diagnostic accuracy of 320-detector CT coronary angiography with prospective and retrospective electrocardiogram (ECG) gating in a single heartbeat. Two independent reviewers separately scored image quality of coronary artery segment for 480 cardiac CT studies in a prospective group and a retrospective group (240 patients with a heart rate <65 beats per minute in each group). The two groups matched well for clinical characteristics and CT parameters. There was good agreement for image quality scores of coronary artery segment between the independent reviewers (κ=0.73). Of the 7023 coronary artery segments, the image quality scores of the prospective group and retrospective group were not significantly different (p>0.05). The mean radiation dose was 10.0 ± 3.5 mSv (range 6.2-21.6 mSv) for prospective ECG gating at 65-85% of R-R interval (the interval between the R-wave of one heartbeat to the R-wave of the next). The mean radiation dose for retrospective ECG-triggered modulated scans was 23.2 ± 3.4 mSv (range 17-27.4 mSv). The mean radiation dose was 57% lower for prospective gating than for retrospective gating (p<0.01). Compared with coronary angiography, the results for prospective vs retrospective ECG gating were 92% vs 90% for sensitivity (p=0.23), 89% vs 91% for specificity (p=0.19), 90% vs 93% for positive predictive value (p=0.25) and 92% vs 95% for negative predictive value (p=0.21) for lesions with ≥50% stenosis, respectively. 320-detector CT coronary angiography performed with prospective ECG gating has similar subjective image quality scores, but a 57% lower radiation dose than retrospective ECG gating in a single heartbeat.