A series of N,N-dimethylated and N-monomethylated analogues of N,N-dimethyl-2-(2'-amino-4'-iodophenylthio)benzylamine substituted at the 4'-phenyl position have been prepared and evaluated in vitro for serotonin transporter (SERT) selectivity. Several derivatives were prepared where the 4'-position was either unsubstituted 13 and 33a or substituted with methyl 14a and 33b, ethenyl 14b and 34, ethyl 16 and 35, hydroxymethyl 20 and 41, hydroxyethyl 22, fluoroethyl 23, hydroxypropyl 27, and fluoropropyl 28. Competition binding in cells stably expressing the transfected human SERT, dopamine transporter (DAT), and norepinephrine transporter (NET) using [(3)H]citalopram, [(3)H]WIN 35,428 or [(125)I]RTI-55, and [(3)H]nisoxetine, respectively, demonstrated the following order of SERT affinity (K(i) (nM)): 14a (0.25) > 16 (0.49) > 20 (0.57) > 14b (1.12) > 13 (1.59) > 33b (1.94) = 35 (2.04) >> 23 (8.50) = 28 (8.55) > 41 (15.11) >> 22 (51) > 33a (83.43) > 27 (92). The K(i) values revealed that most of these derivatives displayed a high affinity for the SERT and a high selectivity over the DAT and NET. Moreover, substitution at the 4'-position of the dimethylated and monomethylated benzylamines differently influenced SERT binding: (i) the dimethylated benzylamines exhibited higher SERT affinity than the monomethylated ones, (ii) alkyl, alkenyl, or hydroxymethyl functions at the 4'-position afford compounds with high SERT affinity, and (iii) omega-hydroxy and fluoro-substituted ethyl and propyl groups at the 4'-position decrease the SERT affinity. From this series, the dimethylated derivatives 13, 14a, 14b, 16, and 20 were radiolabeled with carbon-11 and their log P(7.4) was calculated as a measure of their potential brain penetrance as positron emission tomography SERT imaging agents.