A key need for dynamic single-cell measurements is the ability to gently position cells for repeated measurements without perturbing their behavior. We describe a new method that uses a gentle secondary flow to trap and suspend single cells, including motile cells, at predictable locations in 3-D. Trapped cells can be more dense or less dense than the surrounding medium. The cells are suspended without surface contact in one of four steady streaming eddies created by audible-frequency fluid oscillation (< or =1000 Hz) in a microchannel containing a single fixed cylinder (radius = 125 microm). Comparison of measured trap locations to computations of the eddy flow show that each trap is located near the eddy center, and the location is controlled via the oscillation frequency. We use the motile phytoplankton cell (Prorocentrum micans) to experimentally measure the trapping force, which is controlled via the oscillation amplitude. Trapping forces up to 30 pN are generated while exerting moderate shear stresses (shear stresses < or = 1.5 N/m2) on the trapped cell. The magnitude of this trapping force is comparable to that of optical tweezers or dielectrophoretic traps, without requiring an external field outside the physiological range for cells (the shear stresses are comparable to those found in arterial blood flow). The unique combination of predictable 3-D positioning, insensitivity to cell and medium properties, strong adjustable trapping forces, and a gentle fluid environment makes hydrodynamic tweezers a promising new option for noncontact trapping of single cells in suspension.