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Determination of nuclear DNA content by flow cytometry requires comparison with a reference standard. The use of external standards such as lymphocytes or granulocytes is time‐consuming and inaccurate. Chicken red blood cells (CRBC) have a DNA content of 35% of the human diploid value and have been widely used as internal standard. The ratio calculated on the basis of the peak channel numbers of the standard and the sample and used to indicate the DNA content (DNA ratio) is, however, very sensitive to changes in the zero level adjustment of the flow cytometer. If two internal standards are used the DNA ratio becomes independent of the zero level. Rainbow trout red blood cells (TRBC) have a DNA content of 80% of human diploid cells. A mixture of CRBC and TRBC was prepared and stored in small aliquots at —80°C. This mixture was added to the sample before staining. The day‐to‐day variation …