Tc-tetrofosmin seems to show a prolonged thyroid retention when compared with Tc-sestamibi; this may explain its poorer performance in the dual-phase parathyroid scintigraphy. In order to verify objectively whether and to what extent Tc-sestamibi and Tc-tetrofosmin uptake and retention differ in the thyroid gland, we performed a head-to-head comparison between the tracers in 86 euthyroid subjects enrolled in five centres. Data were compared with those of Tc-pertechnetate obtained from the same subjects. For comparison purposes, quantitative data from the salivary glands were also obtained. Tc-sestamibi showed a higher early thyroid uptake (2.26+/-0.52 vs. 2.01+/-0.49, respectively; P<0.002) and a lower retention (1.4+/-0.37 vs. 1.65+/-0.58, respectively; P<0.002) when compared with Tc-tetrofosmin. This finding provides an objective basis for the poorer results of Tc-tetrofosmin in dual-phase parathyroid scintigraphy. Delayed Tc-tetrofosmin salivary gland uptake was unexpectedly high and approached the Tc-pertechnetate value (2.29+/-0.56 vs. 2.46+/-0.75, respectively; P =not significant). This finding should be kept in mind in order to avoid interpretation pitfalls in thyroid and parathyroid imaging with Tc-tetrofosmin, as well as in other oncological imaging of the neck region. This study definitely establishes that Tc-tetrofosmin and Tc-sestamibi are not twin but rather sibling tracers.