Background: Rats (Rattus norvegicus) are increasingly kept as pets, thus more and more requiring veterinary care. Important differences exist between pet and laboratory rats, but little information is available on pet rats. Diagnosis and treatment of pet rat diseases would benefit from RIs specific for this animal population. Objectives: The aim was to establish RIs for biochemical blood variables, PCV, and body temperature in pet rats. Additionally, influences of sex and feed rationing method (ad libitum, restricted) on these variables were determined. Methods: American Society for Veterinary Clinical Pathology (ASVCP) recommendations were followed. Biochemical variables were measured using an automated chemistry analyzer, the VetScan VS2. Nonparametric statistical methods were used to determine RIs and effects of sex and feed rationing method. Results: Reference intervals were established using values of 123 clinically healthy pet rats (except for globulins and albumin/globulin ratio, n = 120) and were: glucose 6.6-13.7 mmol/L, total proteins 66-88 g/L, albumin 38 g/L, globulins 12-35 g/L, albumin/globulin ratio 1.0-4.7, urea nitrogen 2.5-6.6 mmol/L, creatinine 53 mol/L, total bilirubin 4-7 mol/L, ALP 40-442 IU/L, ALT 22-137 IU/L, amylase 502-1113 IU/L, sodium 133-144 mmol/L, potassium 3.6-5.3 mmol/L, calcium 9.5-10.9 mg/dL, phosphorus 2.3-7.0 mg/dL, PCV 40-50%, and body temperature 35.8-39.3 degrees C. Sex significantly affected 10 variables. No significant influence was found with feed limitation Conclusions: Reference intervals reported in this study will be useful for interpretation of biochemistry analysis in pet rats and therefore improve pet rat medicine.