- Data regarding the atrial electrophysiology of rodents is hard to obtain. We hypothesized that the latency to the first R following programmed S1–S2 stimulation of the atria (defined as RRS2) is affected by atrial capture and may be utilized to determine the atrial refractoriness of rodents using a simple electrocardiogram (ECG)-based algorithm. ECG signals during programmed-stimulation protocols were evaluated in 24 anesthetized rats and 12 mice using an automated QRS detection program. In each S1–S2 stimulation the atrial capture was determined in the invasive recording and the RRS2 was determined independently in the ECG recording. Based on our hypothesis an algorithm was designed to determine atrial capture noninvasively and 95% confidence interval (95% CI) was calculated for the obtained specificity and sensitivity. In rats the ratio between RRS2 and the spontaneous RR interval (RRspon) could identify two decision-relevant ranges: RRS2/RRspon < 1.025 and RRS2/RRspon > 1.113; both indicated capture of S2. In contrast, RRS2/RRspon between these values indicated failure of atrial capture. This algorithm reached sensitivity of 97.7% (95% CI 96.0–99.4%) and specificity of 96.8% (95% CI 94.8–98.8%). Following rapid atrial pacing the same algorithm reached sensitivity of 94.96% (95% CI 92.0–97.9%) and specificity of 99% (95% CI 97.7–100%). In mice, difficulties in QRS detection were encountered which somewhat limited the ECG analysis. Our results indicate that atrial electrical capture affects the latency to the first R following S1–S2 stimulation in a predictable manner. This finding may advance the ability to detect the atrial refractoriness of rodents using a simple, minimally invasive, experimental setting.