A job analysis was conducted to define knowledge important for entry-level middle school science teachers. The results will be used to guide the development of new test content specifications for The Praxis Series™ Middle School Science assessment. A draft domain was constructed from existing test specifications, national standards, and feedback from item writers and reviewers. A national advisory committee (NAC) of middle school science teachers and college faculty reviewed and modified the draft domain to reflect the knowledge they believed was important for safe and effective practice and needed at time of entry into the profession. The revised domain consisted of 72 statements clustered within six major categories: (a) Scientific Inquiry, Methodology, Techniques, and History; (b) Life Sciences; (c) Basic Principles of Matter and Energy; (d) Physical Sciences; (e) Earth and Space Sciences; and (f) Science, Technology, and Society. Survey responses of 435 middle school science educators provided independent evidence of the importance of the domain. Respondents judged each knowledge statement on a scale ranging from 1 (not at all important) to 5 (extremely important). Sixty-one knowledge statements had mean ratings above 3.50 in the aggregate sample and for subgroups containing more than 30 respondents. The results indicate that these 61 statements may be considered appropriate for guiding the development of new test content specifications for the Praxis Middle School Science assessment.