The main purpose of the Ontario Benthos Biomonitoring Network (OBBN) is to enable assessment of aquatic ecosystem condition using benthos as indicators of water and habitat quality. This manual is a companion to the OBBN Terms of Reference, which detail the networks objectives, deliverables, development schedule, and implementation plan. Herein we outline recommended sampling, sample processing, and analytical procedures for the OBBN.The reference condition approach (RCA), which compares the benthic community at test sites (where biological condition is in question) to that of multiple minimally-impacted reference sites, is a commonly used bioassessment study design. Because benthic community composition is determined in large part by environmental attributes (e.g., catchment size, substrate type), a combination of catchment- and site-scale habitat measures are used to select appropriate reference sites that are used to develop bioassessment criteria. A variety of minimally impacted sites must be sampled in order to permit evaluation of the wide range of potential test sites inOntario.We detail sampling and sample processing methods for lakes, streams, and wetlands.Recommended sampling methods define sampling units, and specify sampling effort, replication, collection procedures (e.g., Travelling-Kick-and-Sweep), mesh size, and sampling periods throughout the year. Sample-processing recommendations cover sub-sampling methods, picking methods, detail (taxonomic level) of benthos identification, and sample archiving. Protocols are consistent with a rapid-bioassessment approach. We have tried to strike a balance between protocol standardization and flexibility. Standardization is important to allow comparisons between sites and times, to facilitate data sharing among network participants, and to permit development of quality control procedures. Flexibility is important to allow participation by partners having different amounts of expertise, time, and money, and to allow protocols to be used in studies aimed at answering different questions.In our analysis section, we discuss the need to select appropriate reference sites using suitable predictors of biological community composition. We then describe methods for testing our bioassessment null hypothesis, that the test site is normal (or in reference condition). The biological condition of both the test site and reference sites are first summarized using a set of indices (e.g., percent mayflies, site-score from 1st axis extracted in a correspondence analysis). Using calculations easily performed in Microsoft Excel, we then apply Test Site Analysis (TSA) to convert the values of the selected indices into a single number (a multivariate distance measure). We statistically compare this distance measure to the distances among reference sites to determine how unusual the test site is.Several appendices provide supplementary information: a list of diagnostic characters for the benthos groups comprising our minimum acceptable level of taxonomic resolution, a checklist of families, blank field and taxa enumeration sheets, a list of catchment-scale habitat variables for characterizing sites, OBBN research priorities, a paper explaining how to do TSA in Microsoft Excel, and an equipment checklist. All protocols are subject to testing and refinement in subsequent editions.