PURPOSE: We compared the reproducibility of 201Tl and 99mTc-sestamibi (MIBI) gated SPECT measurement of myocardial function using the Germano algorithm. MATERIALS AND METHODS: Gated SPECT acquisition was repeated in the same position in 30 patients who received 201Tl and in 26 who received 99mTc-MIBI. The quantification of end-diastolic volume (EDV), end-systolic volume (ESV), and ejection fraction (EF) on 201Tl and 99mTc-MIBI gated SPECT was processed independently using Cedars quantitative gated SPECT software. The reproducibility of the assessment of myocardial function on 201Tl gated SPECT was compared with that of 99mTc-MIBI gated SPECT. RESULTS: Correlation between the two measurements for volumes and EF was excellent by the repeated gated SPECT studies of 201Tl (r=0.928 to 0.986; p<0.05) and 99mTc-MIBI (r=0.979 to 0.997; p<0.05). However, Bland Altman analysis revealed the 95% limits of agreement (2 SD) for volumes and EF were tighter by repeated 99mTc-MIBI gated SPECT (EDV: 14.1 ml, ESV: 9.4 ml and EF: 5.5%) than by repeated 201Tl gated SPECT (EDV: 24.1 ml, ESV: 18.6 ml and EF: 10.3%). The root mean square (RMS) values of the coefficient of variation (CV) for volumes and EFs were smaller by repeated 99mTc-MIBI gated SPECT (EDV: 2.1 ml, ESV: 2.7 ml and EF: 2.3%) than by repeated 201Tl gated SPECT (EDV: 3.2 ml, ESV: 3.5 ml and EF: 5.2%). CONCLUSION: 99mTc-MIBI provides more reproducible volumes and EF than 201Tl on repeated acquisition gated SPECT. 99mTc-MIBI gated SPECT is the preferable method for the clinical monitoring of myocardial function.