The purposes of this manual are to: 1) document procedures that were developed and used by the United States Environmental Protection Agencys (USEPA) Ecosystem Exposure Research Division (EERD) for the assessment of the physical and biological characteristics of headwater streams; and 2) provide a catalog of procedures to other groups with an interest in headwater stream assessment. Earlier EPA field operations manuals for running waters have focused on larger systems, including wadeable streams, non-wadeable rivers, and Great Rivers (e.g., Barbour et al. 1999, Lazorchak et al. 1998, 2000, Angradi 2006). There is a growing interest in headwater streams because human activities (e.g., road building, stormwater management) frequently intersect these widespread waterbodies. There is also considerable legal debate regarding extent of jurisdictional waters under the Clean Water Act and the role or nexus of various types of headwater streams to the integrity of downstream interstate waters (Nadeau and Rains in press). Some states, like North Carolina and Ohio, have already began to initiate headwater stream classification methods for regulatory purposes (Ohio Environmental Protection Agency 2002, N.C. Division of Water Quality 2005).