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Choline transport in erythrocytes is irreversibly inhibited by N-ethylmaleimide. The hypothesis that the carrier alternates between outward-facing and inward-facing forms and that only the latter reacts with the inhibitor (Martin, K. (1971) J. Physiol. (London) 213:647--667; Edwards, P.A. (1973) Biochim. Biophys. Acta 311:123--140) is here subjected to a quantitative test. In this test the effects of a series of substrate analogs upon rates of inactivation and rates of choline exit are compared. By hypothesis the effect of an analog in the external solution on the inactivation rate depends only on how it affects the proportion of the inward-facing carrier. Since 14C-choline efflux is necessarily proportional to the concentration of free carrier in the inward-facing form, the analogs should have related effects on the two rates. In every case the observed effects were identical, whether the analogs accelerated transport or inhibited it. Analysis of the results demonstrates that (1) the transport mechanism depends on the operation of a mobile element; (2) distinguishable inward-facing and outward-facing conformations of the free carrier, carrier-substrate complex, and carrier-inhibitor complex exist, and only the inward-facing forms react at a significant rate with N-ethylmaleimide; (3) carrier mechanisms involving a single form of free carrier or a single form of carrier-substrate complex are ruled out; and (4) dissociation of the carrier-substrate complex is a rapid step with all substrate analogs.

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