The purpose of this prospective study was to evaluate the relationship between thallium-201 chloride (201Tl) and technetium-99m hexakis 2-methoxyisobutylisonitrile (99Tcm-MIBI) accumulation and histopathological differentiation in primary lung adenocarcinoma. A total of 43 patients with primary lung adenocarcinoma were investigated. The patients were divided into well differentiated (n = 17), moderately differentiated (n = 14) and poorly differentiated (n = 12) carcinoma groups. Simultaneous dual single photon emission tomography (SPET) images with 201Tl and 99Tcm-MIBI were acquired 15 min (early) and 2 h (delayed) after injection. Using a region of interest technique, the tumour-to-normal lung ratio was calculated for both early (early ratio) and delayed (delayed ratio) images. The retention index was calculated using the formula delayed ratio/early ratio. Uptake of the radionuclides was compared with the classification of tumour differentiation grading. The mean (+/- SD) values of the early ratio, delayed ratio and retention index using 201Tl were 2.19+/-0.72, 2.28+/-0.71 and 1.06+/-0.16, respectively, in the well differentiated group, 2.38+/-0.83, 2.48+/-0.84 and 1.08+/-0.23, respectively, in the moderately differentiated group, and 2.87+/-0.75, 3.60+/-1.51 and 1.22+/-0.21, respectively, in the poorly differentiated group. Both the ratios and the retention index using 201Tl were significantly lower in the well differentiated group than in the poorly differentiated group. The delayed ratio using 201Tl in the moderately differentiated group was also significantly lower than that in the poorly differentiated group. There were no significant differences in either ratio or the retention index among the three groups using 99Tcm-MIBI. 201Tl SPET is superior to 99Tcm-MIBI SPET for the grading of histopathological differentiation of primary lung adenocarcinoma.