One of the most pressing concerns associated with conservation of the endangered Florida manatee is mortality and serious injury due to collisions with watercraft. Watercraft collisions are the leading identified cause of manatee mortality, averaging 25% and reaching 31% of deaths each year. The successful establishment and management of protected areas depend upon the acquisition of data assessing how manatees use different habitats, and identification of environmental characteristics influencing manatee behavior and habitat selection. Acoustic playback experiments were conducted to assess the behavioral responses of manatees to watercraft approaches. Playback stimuli made from prerecorded watercraft approaches were constructed to simulate a vessel approach to approximately 10 m in sea grass habitats. Stimulus categories were (1) silent control, (2) approach with outboard at idle speed, (3) vessel approach at planning speed, and (4) fast personal watercraft approach. Analyses of swim speed, changes in behavioral state, and respiration rate indicate that the animals responded differentially to the playback categories. The most pronounced responses, relative to the controls, were elicited by personal watercraft. Quantitative documentation of response during playbacks provides data that may be used as the basis for future models to predict the impact of specific human activities on manatees and other marine mammal populations.