Reference data files support the evaluation of myocardial perfusion single-photon emission tomography (SPET). The aim of this study was to create a large reference data base for technetium-99m sestamibi SPET, age and gender matched to the general patient population. One hundred and twenty-eight healthy volunteers (76 males and 52 females) with a likelihood of coronary artery disease of less than 5% underwent rest and maximal exercise 99mTc-sestamibi SPET with a 2-day protocol and 180 degrees elliptical rotation. The normalized activity values of 99mTc-sestamibi in the inferior wall differed significantly between men and women. Age variations were found for men in the anterior wall. Normalized activity values in all four walls were strikingly similar during rest and stress. Our results suggest that the use of reference files in 99mTc-sestamibi SPET requires a gender- and, for males, possibly an age-matched reference population. Different reference files at rest and during stress might not be necessary.