The aim of this study was to develop a useful method for obtaining viable tissue samples for establishing cell cultures from skin biopsies of free-ranging cetaceans. The skin biopsies were performed by two methods: dart from an air gun and dart from a crossbow. The dart tip was modified to collect tissue. The tissue was kept in tissue culture medium at ambient temperature, then processed within 24 h. Many modifications in culture technique, with respect to conventional culture methods for human fibroblasts, were made. The cultures thus obtained can be used for many purposes, including genetic and toxicological studies. In toxicology they are an alternative in vitro system for studying threatened animals such as marine mammals. In particular, fibroblasts can be used to test the vulnerability of cetaceans and pinnipeds to different environmental contaminants such as organochlorine compounds, heavy metals and polycyclic aromatic hydrocarbons.