To evaluate structure/function relationships among neurotransmitter transporters, chimeric cDNAs were formed between antidepressant- and cocaine-sensitive norepinephrine and serotonin transporters. To eliminate experimenter bias in the positioning of chimeric junctions, we utilized a novel method that involves the in situ formation of chimeras in transformed Escherichia coli from linearized plasmids that bear a single copy of both parental transporter cDNAs. Colonies recovered after growth on selective media frequently contain plasmids bearing chimeric inserts. Coupled with the vaccinia-T7 expression system, this method allowed us to rapidly generate and evaluate multiple transporter chimeras without the need to introduce compatible restriction sites or the time and expense involved in formation of individual chimeric cDNAs. Transporter chimeras with switch points proximal but not distal to the middle of putative transmembrane domain 1 retain serotonin or norepinephrine transport and high-affinity antagonist recognition. Loss of substrate and antagonist recognition despite normal levels of transporter protein by distal chimeras suggests important and divergent interactions between multiple transmembrane domains in forming ligand binding sites. The method of chimera synthesis applied in these studies is likely to be of generic utility particularly when the formation of a library of chimeric cDNAs is desired.