Introduction: Melanocytes of the membranous labyrinth of the inner ear have been described morphologically in various contexts. Nature and functions of these cells are as yet not completely clear, even though several hypotheses exist regarding the same. The limited knowledge is due in part to a lack of methods regarding in vitro cell culture. The aim of this study was to describe conditions for the successful cell culture of vestibular inner ear melanocytes (VIEM), to compare their growth properties with those of epidermal melanocytes, and to characterize them immunohistochemically. Materials and Methods: Membranous labyrinth cells from freshly slaughtered sheep were isolated, and melanocytes and fibroblasts subsequently cultured. In addition, melanocytes from the skin of the same sheep were cultured. Antibodies specific to tyrosinase, tyrosinase-related protein 1 (TRP-1/Mel-5), and melanoma-specific antigen A (Melan A) were used to analyze the cultured cells. Results: The proliferation of VIEM was retarded in comparison to epidermal melanocytes. After 14 days, VIEM began to proliferate for the first time, whereas epidermal melanocytes proliferated already after 7 days. In contrast to epidermal melanocytes, the culturing process of VIEM seemed to be dependent on the presence of fibroblasts, and VIEM often accumulated in the vicinity of fibroblasts forming three-dimensional clusters. Moreover, VIEM showed a higher ratio of highly pigmented cells with a round cell shape and small dendrites in comparison to epidermal melanocytes. Immunohistochemical techniques proved the VIEM to be positive for Melan A, TRP-1 and, in the majority of cases, also for tyrosinase. Conclusion: We successfully cultured melanocytes of the inner ear vestibular labyrinth for the first time and demonstrated melanocytic characteristics of these cells. This accomplishment will provide the opportunity to investigate VIEM in more detail in future experiments.