To assess targeting of an epothilone folate conjugate (BMS-753493) to the folate receptor (FR)-overexpressed tumor in mice bearing both FR+ and FR- tumors, a series of experiments were conducted by quantitative whole-body autoradiography (QWBA) and LC-MS/MS following i.v. administration of BMS-753493 or its active moiety, BMS-748285 in mice bearing FR+ (98M109) and FR- (M109) tumors. QWBA showed [H]BMS-753493-derived radioactivity was extensively distributed to various tissues. The FR over-expressing 98M109 tumors showed consistently higher level of radioactivity than FR-negative tumors (., M109 tumors) up to 48 h post dose of [H]BMS-753493, despite the magnitude of difference between the tumors is relatively small (generally 3~5-fold). The radioactivity level in 98M109 tumors was 2~12-fold of normal tissues except intestine/content at 48 h post dose. No selective radioactivity uptake into 98M109 tumors over M109 or normal tissues was observed after i.v. administration of the active epothilone, [H]BMS-748285. LC-MS/MS measurements demonstrated that the concentrations of BMS-748285, presumably from hydrolysis of the folate conjugate, in 98M109 tumors were greater than those in M109 tumors after i.v. administration of BMS-753493 (2-3-fold) whereas no differential uptake in the tumors following BMS-748285 administration. Those data were consistent with radioactivity determinations. Those results demonstrated that the folate conjugation in BMS-753493 enabled moderately preferential distribution of the active epothilone to FR over-expressing 98M109 tumors, thereby supporting targeted delivery of cytotoxics through the folate receptor.