The activity of the serotonin transporter of platelet plasma membrane may be assessed by its ability to bind [N-methyl-3H]imipramine, even in the presence of detergents which disrupt the membrane and inactivate serotonin transport. Of the 21 detergents which were tested for their ability to solubilize the transporter in an active form, some, such as Triton X-100, inactivate imipramine-binding activity in a manner which is difficult or impossible to reverse, while inactivation by others, such as cholate, is readily reversed by gel filtration. Of the detergents which do not inactivate imipramine binding, most, such as the Tween series, also fail to solubilize significant amounts of the transporter. Of all the detergents tested, only one, digitonin, solubilized specific imipramine-binding activity. Imipramine binds to the digitonin-solubilized transporter with slightly reduced affinity, but in all other respects binding to intact and digitonin-solubilized membranes is identical. Thus, binding is, in both cases, saturable and Na+-dependent, and is reversed by serotonin. Furthermore, inhibitors of serotonin transport but not of serotonin-mediated platelet aggregation block imipramine binding to the soluble preparation. Sodium-dependent binding to the soluble extract is insensitive to K+, suggesting that separate sites on the serotonin transporter mediate the effects of effects of Na+ and K+.