This document describes a process for seeking EPA approval of microbiological alternate test procedures (ATPs) or new methods for use in monitoring drinking water, ambient water, and wastewater. This document serves as a supplement to the ATP guidelines at 40 CFR 136.4, 136.5, and 141.27. With this guidance document, EPA has revised and combined the three existing microbiological ATP protocols to make the ATP process more transparent to applicants while maintaining the same level of data quality. The guidelines in Quantitative Membrane Filter Methods (Reference 10.16), Presence/Absence Liquid Culture Methods for Finished Water (Reference 10.17), and Presence/Absence Membrane Filter Methods for Finished Waters (Reference 10.15) have been incorporated into this single protocol, which is applicable to a wider range of analytes and techniques. This revised ATP protocol describes a process for conducting side-by-side method comparisons and for conducting quality control (QC) acceptance criteria-based method studies for EPA-approved reference methods with QC acceptance criteria. Additionally, in some cases the revised protocol provides applicants an opportunity to demonstrate comparability by meeting QC acceptance criteria associated with the EPA-approved reference methods for different combinations of analyte and determinative technique.