In this study, human embryonic kidney (HEK)-293 cells stably expressing human, Drosophila, or a chimeric serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT, dSERT, and H(1-281)D(282-476)H(477-638), respectively) were used to explore the ability of two libraries of structurally distinct psychostimulants to inhibit 5-HT uptake. One library consisted of 3-phenyltropane analogs, whereas the second library consisted of several substituted amphetamines. hSERT exhibited a lower K(i) value for all the compounds in both libraries compared with dSERT, whereas the chimeric SERT exhibited properties more closely resembling those of dSERT. This species selectivity was explored using computer-generated comparative molecular field analysis to model the interactions of the cocaine analogs and substituted amphetamines at hSERT, dSERT, and the cross-species chimera. Models for the 3-phenyltropane analogs indicate that a region exists around the aromatic ring where decreased electron density is favored, particularly for hSERT. This finding may indicate pi-pi stacking with an aromatic amino acid residue in SERT. Also, electronegative substituents in the 4'-position provide favorable interactions. This structural feature was demonstrated by increased potency of analogs with electronegative substituents on the aromatic ring that withdraw electron density. For the substituted amphetamines, key areas for interaction exist around the amine, an electrostatic component surrounding the 3-position on the aromatic ring, and a steric component surrounding the 4-position.