Vesicular membrane trafficking during acrosome biogenesis in bull and rhesus monkey spermatogenesis differs from the somatic cell paradigm as imaged dynamically using the Golgi apparatus probes β-COP, giantin, Golgin-97, and Golgin-95/GM130. In particular, sorting and delivery of proteins seemed less precise during spermatogenesis. In early stages of spermiogenesis, many Golgi resident proteins and specific acrosomal markers were present in the acrosome. Trafficking in both round and elongating spermatids was similar to what has been described for somatic cells, as judged by the kinetics of Golgi protein incorporation into endoplasmic reticulum-like structures after brefeldin A treatment. These Golgi components were retrieved from the acrosome at later stages of differentiation and were completely devoid of immature spermatozoa. Our data suggest that active anterograde and retrograde vesicular transport trafficking pathways, involving both β-COP- and clathrin-coated vesicles, are involved in retrieving Golgi proteins missorted to the acrosome and in controlling the growth and shape of this organelle.