A new series of compounds related to the nucleoside transport inhibitors, lidoflazine and mioflazine, is introduced. The influence of these derivatives on nucleoside-specific transport proteins was studied in two ways. First, a rapid, non-radioactive assay was developed for the screening of this type of material for actual transport inhibition in human erythrocytes. The method is based on the dose-dependent reversal of the inhibition of inorganic phosphate release induced by inosine when human erythrocytes are suspended in a phosphate-free medium. It enables the estimation of the potency and specificity of this new series of nucleoside transport inhibitors, most of which are highly active (EC50 values as low as 13 nM). Second, the displacement of a radiolabeled transport inhibitor, [3H]nitrobenzylthioinosine, was examined. All compounds were capable of displacing specific [3H]nitrobenzylthioinosine binding to crude and solubilized plasma membranes of calf lung tissue, displaying affinities in the nanomolar range. Pseudo-Hill coefficients derived from the shape of the displacement curves were significantly greater than unity for most derivatives, in contrast to values of approximately unity obtained for dipyridamole and analogs. These findings were incorporated in a mathematical model describing the interaction of mioflazine analogs with the transport protein, suggesting that one molecule of mioflazine is capable of displacing two or more molecules of [3H]nitrobenzylthioinosine at a time. The consequences of this model regarding the nature of the transport protein are discussed.