The kinetic properties of the mediated transport of chloroquine in human erythrocytes are investigated. The high rates of translocation across the cell membrane and high adsorbance properties to glass surfaces have led to the development of new techniques for measuring initial rates of transport. Three different methodological procedures are used to accomplish a complete kinetic characterization of the system. All measurements were done at 25 degrees C. Under zero-trans conditions the system displays complete symmetry, the Michaelis constants being 39.2 +/- 2.4 microM for influx and 36.6 +/- 5.6 microM for efflux. The respective maximal velocities are 206.4 +/- 36.0 microM . min-1 and 190.0 +/- 7.8 microM . min-1. Under equilibrium-exchange conditions the Michaelis constant is 108.6 +/- 15.6 microM and the maximal velocity is 630.3 +/- 50.4 microM . min-1. This 3-fold increase in both K and V over the zero-trans values indicates that the rate-limiting step in the transport of chloroquine is the movement of the unloaded carrier. The kinetic data are consistent with the prediction of a simple carrier model.