Under laminar flow conditions, when no external forces are applied, particles are generally thought to follow fluid streamlines. Contrary to this perspective, we observe that flowing particles migrate across streamlines in a continuous, predictable, and accurate manner in microchannels experiencing laminar flows. The migration is attributed to lift forces on particles that are observed when inertial aspects of the flow become significant. We identified symmetric and asymmetric channel geometries that provide additional inertial forces that bias particular equilibrium positions to create continuous streams of ordered particles precisely positioned in three spatial dimensions. We were able to order particles laterally, within the transverse plane of the channel, with >80-nm accuracy, and longitudinally, in regular chains along the direction of flow. A fourth dimension of rotational alignment was observed for discoidal red blood cells. Unexpectedly, ordering appears to be independent of particle buoyant direction, suggesting only minor centrifugal contributions. Theoretical analysis indicates the physical principles are operational over a range of channel and particle length scales. The ability to differentially order particles of different sizes, continuously, at high rates, and without external forces in microchannels is expected to have a broad range of applications in continuous bioparticle separation, high-throughput cytometry, and large-scale filtration systems.cell manipulation ͉ Dean flow ͉ filtration ͉ hydrodynamic lift ͉ microfluidics C ontinuous manipulation and separation of microparticles, both biological and synthetic, is important for a wide range of applications in industry, biology, and medicine (1-3). Traditional techniques of particle manipulation rely on laminar flow (4) or differences in either particle mobility or equilibrium position in a flow with a variety of externally applied forces (5-7). Recently, microfluidic systems have been shown to be very useful for particle handling with increased control and sensitivity. Systems have been demonstrated that use scale-dependent electromagnetic forces (8-11), microscale hydrodynamic effects (12-14), or deterministic physical interactions and filters (15)(16)(17). However, the precision of microfluidic systems based on deterministic interaction with walls or posts may be limited by disturbances from random interparticle contact and spacing, and mechanical systems are prone to clogging. Additionally, throughput for particle manipulation based on external forces has been limited because the time for forces to act decreases with increasing flow rate. Inertial hydrodynamic forces that would increase along with flow rate have, in all but a few cases (18), not been considered in microfluidic systems. This is because of a widely held notion that small length scales require that the Reynolds number (Re), a measure of the relative importance of inertial to viscous forces, must be concurrently small, precluding any practically useful inertial effects.Inertial...