The normal trans-myocardial tissue hematocrit distribution (i.e., sub-epicardial greater than sub-endocardial) is known to be affected by red blood cell (RBC) aggregation. Prior studies employing the use of infused large macromolecules to increase erythrocyte aggregation are complicated by both increased plasma viscosity and dilution of plasma. Using a new technique to specifically alter aggregation behavior by covalent attachment of Pluronic F98 to the RBC surface, we have determined the effects of only enhanced aggregation (i.e., Pluronic F98-coated RBC) versus enhanced aggregation with increased plasma viscosity (i.e., added 500 kDa dextran) on myocardial tissue hematocrit in rapidly frozen guinea pig hearts. Although both approaches equally increased aggregation, tissue hematocrit profiles differed markedly: a) when using Pluronic F98-coated cells the normal trans-myocardial gradient was abolished; b) when using added dextran the hematocrit remained at sub-epicardial levels for about one-half the thickness of the myocardium, then rapidly decreased to the control level in the sub-endocardial layer. Our results indicate that myocardial hematocrit profiles are sensitive to both RBC aggregation and to changes of plasma viscosity associated with increased RBC aggregation. Further, they suggest the need for additional studies to explore the mechanisms affecting RBC distribution in three-dimensional vascular beds.