The rat Na(+)- and Cl(-)-dependent serotonin transporter was expressed in Sf9 insect cells using the baculovirus system. Expression of the serotonin transporter caused the Sf9 cells to accumulate [3H]serotonin (Km 78 nM) and to bind the specific transport inhibitor [125I]RT155 (2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane) (Kd 0.22 nM). Ligand binding assays on isolated membranes showed 500,000 copies of the serotonin transporter/cell (9 pmol/mg of membrane protein). Immunoreactive bands of apparent M(r) 54,000 (unglycosylated) and 60,000 (glycosylated) were observed in Western blots of membrane proteins from infected cells. The 54-kDa band was significantly smaller than the expected M(r) of 72,500 predicted from the cDNA sequence. The 54-kDa band was shown to represent the intact serotonin transporter by expressing a recombinant serotonin transporter that contained c-Myc and FLAG epitope tags engineered at the N and C termini, respectively. Both tags were present on a membrane protein that migrated slightly slower than the previously observed 54-kDa band, consistent with the extra mass added by the tags. The tags did not affect the Kd for [125I]RT155 binding. The effect of N-linked glycosylation on ligand binding and the level of expression were studied. The expression of the serotonin transporter in tunicamycin-treated Sf9 cells resulted in low levels of ligand binding activity (0.2 pmol/mg) but unchanged Kd. Similarly, mutated serotonin transporters that contained reduced numbers of N-linked glycosylation sites had unchanged Kd for [125I]RT155 binding whether there were 2, 1, or 0 N-linked glycosylation sites present on the serotonin transporter. In contrast, Bmax was dramatically reduced; levels of expression of the unglycosylated serotonin transporter (0.4 pmol/mg) were 20-fold lower compared with levels of the fully glycosylated serotonin transporter. The Km for [3H]serotonin uptake was also unchanged. These data indicate that glycosylation is required for optimal stability of the serotonin transporter in the membrane but not for serotonin transport or ligand binding per se.