The targeting of synaptophysin, a major synaptic vesicle protein, in transfected nonneuronal cells has important implications for synaptic vesicle biogenesis, but has proved controversial. We have analyzed four transfected cell types by differential centrifugation and velocity gradient sedimentation to determine whether synaptophysin is targeted to endosomes or to synaptic vesicle-like structures. Synaptophysin was recovered only in vesicles that sedimented more rapidly than synaptic vesicles. The synaptophysin-containing vesicles were labeled if a surface-labeled cell was warmed to 37 degrees C, comigrated with transferrin receptor-containing vesicles on velocity and density gradients, and could be completely immunoadsorbed by anti-LDL receptor tail antibodies. These data demonstrate that synaptophysin was targeted to the early endocytotic pathway in the transfected cells and are inconsistent with the suggestion that synaptophysin expression induces a novel population of vesicles. Targeting of synaptophysin to early endosomes implicates their role in synaptic vesicle biogenesis.