The ink gland of the cuttlefish Sepia officinalis has traditionally been regarded as a convenient model system for investigating melanogenesis. This gland has been shown to contain a variety of melanogenic enzymes including tyrosinase, a dopachrome-rearranging enzyme and peroxidase. However, whether and to what extent these enzymes co-localize in the melanogenic compartments and interact is an open question. Using polyclonal antibodies that recognize the corresponding Sepia proteins, we have been able to demonstrate that peroxidase has a different subcellular localization pattern from tyrosinase and dopachrome-rearranging enzyme. Whereas peroxidase is located in the rough endoplasmic reticulum and in the matrix of premelanosomes and melanosomes, tyrosinase and dopachrome-rearranging enzyme are present in the rough endoplasmic reticulum-Golgi transport system, at the level of trans-Golgi cisternae, trans-Golgi network and coated vesicles, and in melanosomes on pigmented granules. These results fill a longstanding gap in our knowledge of the melanin-producing system in Sepia and provide the necessary background for dissection at the molecular level of the complex interaction between melanogenic enzymes. Moreover, the peculiar and complex organization of melanin in an invertebrate such as Sepia officinalis is surprising and could provide the basis for understanding the process in more evolved systems such as that of mammals.