SummaryThe effects of three phlebotomy techniques — periorbital sinus puncture, tail vein incision and cardiac puncture — were determined on seven hematological parameters and seven clinical chemical parameters in female Sprague‐Dawley rats. The mean er‐ythrocyte count, leukocyte count, hemoglobin and hematocrit were reduced with cardiac puncture as compared to the other two techniques. There was a statistically significant increase in the variance of each of these parameters except the leukocyte count. The mean serum lactate dehydrogen‐ase, aspartate animotransferase, alanine aminotransferase, gamma‐glutamyl transfer‐ase and creatinine were greater in samples collected by cardiac puncture than with the periorbital sinus and tail vein techniques. A statistically significant difference in variance was observed between the orbital sinus puncture and the cardiac puncture for each of these parameters. In all cases except lactate dehydrogenase, the values from the orbital sinus and tail vein techniques were comparable for both hematology and clinical chemistry. A large variance was found by all three techniques for both alkaline phos‐phatase and lactate dehydrogenase in the rat. Over 60% of the serum samples were hemolyzed from the cardiac puncture technique while approximately 25% of the serum samples from tail vein incision were hemolyzed. In this laboratory the lack of hemolysis and the lower variance make the periorbital sinus venipuncture technique the method of choice for collection of blood sa