This usability study evaluated users' perceptions of a multimedia prototype for a new e-learning tool: Anatomy of the Central Nervous System: A Multimedia Course. Usability testing is a collection of formative evaluation methods that inform the developmental design of e-learning tools to maximize user acceptance, satisfaction, and adoption. Sixty-two study participants piloted the prototype and completed a usability questionnaire designed to measure two usability properties: program need and program applicability. Statistical analyses were used to test the hypothesis that the multimedia prototype was well designed and highly usable, it was perceived as: (1) highly needed across a spectrum of educational contexts, (2) highly applicable in supporting the pedagogical processes of teaching and learning neuroanatomy, and (3) was highly usable by all types of users. Three independent variables represented user differences: level of expertise (faculty vs. student), age, and gender. Analysis of the results supports the research hypotheses that the prototype was designed well for different types of users in various educational contexts and for supporting the pedagogy of neuroanatomy. In addition, the results suggest that the multimedia program will be most useful as a neuroanatomy review tool for health-professions students preparing for licensing or board exams. This study demonstrates the importance of integrating quality properties of usability with principles of human learning during the instructional design process for multimedia products. Anat Sci Ed 2008. © 2008 American Association of Anatomists.