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Converting fluorescence to absolute numbers of molecules requires a fluorescent standard whose molecular composition is known precisely. In this report, the construction, properties and mode of using a set of fluorescence calibration standards are described. The standards are based on an icosahedral virus engineered to contain exactly 240 copies of one of seven different fluorescent proteins. Two applications of the fluorescent standards to counting molecules in the human parasite <jats:italic>Toxoplasma gondii<\/jats:italic> are described. 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