Purpose: The aim of this study was to investigate the intra-individual reproducibility of left ventricular volume and ejection fraction measurements in living rat using pinhole gated single-photon emission computed tomography (SPECT). Methods: Eight normal male Wistar rats underwent four pinhole gated SPECT acquisitions over a 1-month period. Two pinhole gated myocardial perfusion SPECT studies were acquired at a 1-week interval after injecting the animals with 43952 MBq of 99mTc-sestamibi. Subsequently, 1 week after the perfusion studies, two pinhole gated blood pool SPECT studies were acquired at a 1-week interval after in vivo labelling of the red blood cells using 52049 MBq of 99mTc-pertechnetate. Pinhole gated SPECT acquisitions were done on a single-head gamma camera equipped with a pinhole collimator with a 3-mm opening and 165-mm focal length. Parameters of acquisition were as follows: 44 mm radius of rotation, 360 rotation using a circular orbit, 64 projections, 64x64 matrix, gating using 16 time frames and 22-min acquisition time. The projection data were reconstructed with a modified version of OSEM taking into account the pinhole geometry and incorporating a prior assumption about the temporal properties of gated SPECT studies to reduce noise. Left ventricular volumes and ejection fraction were measured using automatic quantification algorithms. Inter-study, inter-observer and intra-observer reproducibility was investigated. Results: Pinhole gated myocardial perfusion and pinhole gated blood pool images were of high quality in all animals. No significant differences were observed between the repeated measurements. The pinhole gated myocardial perfusion SPECT studies indicated that differences between repeated measurements larger than 41 ml for end-diastolic volume, 17 ml for end-systolic volume and 3% for ejection fraction were significant. The pinhole gated blood pool SPECT studies indicated that differences between repeated measurements larger than 42 mul for end-diastolic volume, 38 mul for end-systolic volume and 5% for ejection fraction were significant. In addition to the reproducibility measures, the accuracy of volume measurements in pinhole gated blood pool SPECT was confirmed by a phantom study. Excellent correlations were observed between the measured volumes and the actual phantom volumes. Conclusion: Pinhole gated SPECT is an accurate and reproducible technique for cardiac studies of small animals. Because this technique is non-invasive, the same animal can be imaged repetitively, allowing follow-up studies.