We used atomic force/lateral force microscopy (AFM/LFM) and the transfer of a lipophilic fluorescent dye (octadecylrhodamine G, R-18) to study the morphology and fusion of human prostasomes at pH values ranging between 5 and 8. The AFM data revealed that the mean diameter of untreated prostasomal vesicles was about 200 nm, but a second prostasome population with a diameter of about 400 nm is also present. This second population increased upon exposing the vesicles to low pH values. The AFM/LFM characterization revealed the smoothness of the prostasome surface and showed that the larger vesicles were fused prostasomes, as also indicated by the relief of R-18 self-quenching.