We evaluated lipophilicity, in vitro cell accumulation, and biodistribution of a series of 99mTc-ether isonitrile complexes to determine whether increased lipophilicity promotes extraction by tumor or enhances imaging properties of the radiopharmaceutical. Nine 99mTc-sestamibi analogs were synthesized and their lipophilicity was determined. Net cellular accumulation and membrane-potential-independent uptake were quantitatively compared in cultured human colon, breast, and lung carcinoma cells. The biodistribution of [99mTc-(2-methoxy-2-ethyl-isocyanopropane)6]+ (99mTc-MMBI) and [99mTc-(2-ethoxy-2-methyl-1-isocyanopropane)6]+ (99mTc-EIBI) was studied in nude mice using subcutaneous, subrenal capsule, and hepatic tumor xenografts. Accumulation of these compounds in colon cells correlated with increasing lipophilicity. Compared with 99mTc-sestamibi, 99mTc-EIBI exhibited (i) in colon cells both higher net accumulation and a higher specific/nonspecific uptake ratio; (ii) in all three cell lines higher membrane-potential-dependent accumulation; and (iii) in all subcutaneous tumor xenografts and in colon subrenal capsule and hepatic tumor xenografts higher tumor/background ratios.