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Commonly obtained clear plasma or serum samples contain small amounts of hemoglobin derived from either naturally occurring processes or from collection trauma. Using the recently evaluated, highly specific, sensitive, quantitative procedure of first derivative spectroscopy, the hemoglobin concentration in nine types of vacuum collection containers was measured, including three types of anticoagulant, three tube sizes, and tubes with or without serum separators. Using this procedure, reference values were established for plasma and serum hemoglobin. These values are the result of the combined effect of red cell trauma during the collection, interaction with the anticoagulant or the serum separator material, and physiologic changes. The observed hemoglobin levels were best described by a log normal distribution pattern. The three plasma collection samples showed lower average values than the serum samples. The citrate anticoagulant gave the lowest plasma hemoglobin average value of 32.7 mg per L (3.27 mg per dl) 9.2 to 123 mg per L (0.92 to 12.3 mg per L 2SD range). It is concluded that the average hemoglobin concentration owing to the collection procedure is less than 100 mg per L (10 mg per dl). A serum or plasma hemoglobin concentration over 200 mg per L (20 mg per dl) is probably due to abnormal physiologic causes and not collection artefact. Serum or plasma hemoglobin measurements evaluated by these criteria can be relied upon as an accurate approximation of excess hemoglobin produced by a pathologic hemolytic process such as acute autoimmune hemolytic anemia, major blood group incompatibility or paroxysmal hemoglobinuria.