This data file standard for flow cytometry (FCS) provides a detailed description of a data file structure designed such that the file can include all of the information necessary to describe fully: (1) The instrument used to obtain the data; (2) the sample measured; (3) the data obtained; and (4) the results of analysis of the data. The file may contain one or more data sets. Each data set includes data from a single sample or acquisition run. Each data set consists of a minimum of four parts—HEADER, TEXT, DATA, and ANALYSIS—all of which are required. The file is structured such that the parts of a data set do not have to be in any particular order, except for the HEADER, which must always be first. The HEADER contains pointers to the beginning and end of each of the other three parts. The file is written as a continuous byte stream, with no line or page formatting. The HEADER, TEXT, and ANALYSIS parts are written in ASCII format and the DATA part can be written in ASCII, binary integer or floating point. This Standard, noted as FCS2.0, is based on a standard proposed by Murphy and Chused (Cytometry 5:553–555, 1984). Data written utilizing their proposed format (FCS1.0) is compatible with the format described here. Future versions of the standard will maintain compatibility with older versions.