The use of hemoglobin-based oxygen carrier solutions in patients requiring blood transfusion will necessitate that clinical laboratories have mechanisms in place to evaluate the potential interference effect of these substances on testing methods. Because these oxygen carrier solutions contain acellular hemoglobin, but do not contain many of the intracellular enzymes and ions present in erythrocytes, interference effects from blood substitutes may be quite different when compared to in vivo or in vitro lysis of erythrocytes. We evaluated the potential interference effect of Diaspirin Cross-linked Hemoglobin on 29 different clinical laboratory analytes. Various combinations of these analytes were tested using the Hitachi 747 and 911 systems, a Beckman CX3, an Abbott AxSym, a Bayer Immuno I, and a Dade ACA IV; a total of 60 analyte/instrument combinations. We used the method of multiple regression analysis to classify interferences as analyte-dependent, analyte-independent, or a combination of the first two types. The presence of clinically significant test interference was derived by using the criteria for maximum allowable error specified in the Clinical Laboratory Improvement Amendments of 1988. Using these criteria, we found significant interference from Diaspirin Cross-linked Hemoglobin with 13 of 29 analytes tested. Interference was noted with the Hitachi 747 and 911 methods for albumin, alkaline phosphatase, total and conjugated bilirubin, cholesterol, total carbon dioxide, iron, lactate dehydrogenase, magnesium, total protein, and triglyceride. In addition, Diaspirin Cross-linked Hemoglobin interfered with measurement of L-lactate using the ACA IV and minor interference was noted with glucose measured using the Beckman CX3. Data from the interference studies was graphically displayed in the form of interference plots. These plots show the maximum allowable test error, due to Diaspirin Cross-linked Hemoglobin, as a function of analyte and interferent concentrations. Evaluation of the potential interference effect of hemoglobin-based oxygen carrier solutions with use of multiple regression analysis and graphical display of the resultant data in the form of interference plots allows for more reliable reporting of test results from specimens containing these products.