Separation of red blood cells and plasma in microcirculatory vessels produces a cell-free layer at the wall. This layer may be an important determinant of blood viscosity and wall shear stress in arterioles, where most of the hydraulic pressure loss in the circulatory system occurs and flow regulatory mechanisms are prominent. With the use of a newly developed method, the width of the cell-free layer was rapidly and repeatedly determined in arterioles (10- to 50-microm inner diameter) in the rat cremaster muscle at normal arterial pressure. The temporal variation of the cell-free layer width was non-Gaussian, but calculated mean and median values differed by <0.2 microm. The correlation length of the temporal variations downstream (an indication of mixing) was approximately 30 microm and was independent of pseudoshear rate (ratio of mean velocity to vessel diameter) and of vessel diameter. The cell-free layer width was significantly different on opposite sides of the vessel and inversely related. Increasing red blood cell aggregability reduced this inverse relation but had no effect on correlation length. In the diameter range studied, the mean width of the cell-free layer increased from 0.8 to 3.1 microm and temporal variations increased from 30% to 70% of the mean width. Increased aggregability did not alter either relationship. In summary, the cell-free layer width in arterioles is diameter dependent and shows substantial non-Gaussian temporal variations. The temporal variations increase as diameter increases and are inversely related on opposite sides of the vessel.