Although the effects of red blood cell (RBC) aggregation on low shear rate blood viscosity are well known, its effects on in vivo flow resistance are still not fully resolved. The present study was designed to explore the in vivo effects of RBC aggregation on flow resistance using a novel technique to enhance aggregation: cells are covalently coated with a block copolymer (Pluronic F98), then suspended in unaltered plasma. RBC aggregation was increased in graded steps by varying the Pluronic concentration during cell coating, and was verified by microscopy and by the erythrocyte sedimentation rate (ESR); the rate increased by 200% at the highest Pluronic level. RBC suspensions were perfused through an isolated in situ guinea pig hind limb preparation, with the arterial perfusion pressure held constant at 100 mmHg via a pressure servo-controlled pump. No significant effects of enhanced RBC aggregation were observed when studies were conducted in preparations with intact vascular control mechanisms. However, after inhibition of smooth muscle tone (10^-4 M papaverin), a significant increment in flow resistance was observed for RBC suspension having a 97% increase of ESR. Further enhancements of RBC aggregation (i.e., 136% and 162% increase of ESR) decreased flow resistance almost to the control values. This was followed by another significant increase in flow resistance during perfusion with RBC suspensions having a 200% increase of ESR. This tri-phasic effect of graded increases of RBC aggregation is most likely explained by an interplay of several hemodynamic mechanisms that are triggered by enhanced RBC aggregation.