Storage of 5-hydroxytryptamine (5HT) in membrane-bound vesicles (dense bodies) of platelets has been proposed to occur as a result of the formation of macromolecular complexes between nucleotides and 5HT, or because of the existence of an electrochemical proton gradient (delta mu H+) across the vesicle membrane. Tests of the applicability of these hypotheses to 5HT storage in the dense bodies of human platelets have been made by examining the disposition of quinacrine and 5HT in these organelles following varying treatments. Binding seems unlikely, since solid analogues of the dense body core (calcium, adenine nucleotides, and pyrophosphate) do not significantly bind 5HT or quinacrine. Incubation of platelets with substances which disrupt delta mu H+ releases a large percentage of the intra-platelet quinacrine. A much smaller fraction of the total platelet 5HT is released by similar treatment, suggesting that the delta mu H+ may not be required for 5HT storage. Because inhibition of the de novo uptake of 5HT into dense bodies fails to cause the significant loss of the 5HT stored in this compartment, 5HT stores do not appear to be maintained by active 5HT uptake. Several substances which enter the dense bodies equally well at 0 degrees C and 37 degrees C cause release of 5HT at 37 degrees C but not at 0 degrees C. The release observed at 37 degrees C thus cannot be attributable to collapse of delta mu H+ or to the displacement of 5HT from intra-granular binding sites, but may be related to increased membrane permeability to 5HT at 37 degrees C. Based on these observations, it appears as if 5HT taken up into the dense bodies of human platelets is retained because the dense body membrane has a very low passive permeability for 5HT, and that many compounds which cause 5HT release at 37 degrees C may act by increasing this permeability.