Assessment and management of Atlantic bluefin tuna Thunnus thynnus populations is hindered by our lack of knowledge regarding trans-Atlantic movement and connectivity of eastern and western populations. Here, we evaluated migratory and homing behaviors of bluefin tuna in several regions of the North Atlantic Ocean and Mediterranean Sea using chemical tags (delta C-13 and delta O-18) in otoliths. Significant emigration of bluefin tuna from their place of origin was inferred from otolith delta C-13 and delta O-18, with both eastern and western bluefin tuna commonly 'crossing the line' (45 degrees W management boundary) in the Central North Atlantic Ocean and mixing with the other population. Several western migrants were also detected in Moroccan traps off the coast of Africa, indicating that trans-Atlantic movement occurs for members of the western population; however, the degree of mixing declined with proximity to the eastern spawning area (Mediterranean Sea). The origin of bluefin tuna collected at the entrance to the Strait of Gibraltar and from several regions within the Mediterranean Sea (Balearic Islands, Malta, and Sardinia) was essentially 100% eastern fish, demonstrating that natal homing is well developed by the eastern population, with western migrants rarely entering the Mediterranean Sea.