Clinical chemistry data are decisive for evaluating altered organ function or damage in experimental animals. Few publications provide reliable clinical chemistry reference intervals, and analytical methods are often not described. Here, we investigated common clinical chemistry values in adult male and female Wistar rats and C57/BL6 mice (n=30/group). Blood samples were taken and analysed for electrolytes, substrates, metabolites and enzymes. In addition, we investigated cystatin C, an important marker of glomerular dysfunction. All data were obtained using commercially available kits frequently employed in most clinical chemistry laboratories and compared with data from other studies, as well as with human data. Significant gender-specific differences were observed in rats (electrolytes, retention parameters and transaminases) and in mice (cholesterol, glucose). High variability was noted for sodium, potassium, glucose, creatine kinase, lactate dehydrogenase and transaminase levels. Both rodent species showed markedly higher alpha-amylase activity than humans. This report demonstrates significant differences between genders for many analytes in rats and for fewer parameters in mice. Some reference values displayed major discrepancies between rodents and humans.