Previous studies showed that amantadine transport increased while tetraethylammonium (TEA) transport decreased in kidney tissue from diabetic rats. Changes in transport activity were reversed by exogenous insulin. We hypothesized that this difference in transport regulation is due to differential regulation of different transport systems. Native human embryonic kidney cortex cells (HEK293 cell line) and rat organic cation transporter (rOCT)-transfected cells were used to test the hypothesis. In support of differential regulation, short-term glucose starvation stimulated amantadine transport and inhibited TEA transport, but the effect was bicarbonate-modulated only for amantadine. cAMP analogues inhibited TEA transport while stimulating amantadine transport. This effect was additive to the effect of insulin, and the presence of bicarbonate affected the extent of the change. Our findings indicated that regulation of rOCT 1 and 2 was mediated by transmembrane adenylyl cyclase, and regulation of amantadine transport was mediated by soluble adenylyl cyclase, suggesting that intracellular microdomains of cAMP may be important in determining overall cellular transport for organic cations. Soluble adenylyl cyclase activity is known to be modulated by bicarbonate and lactate. These observations support our hypothesis and reconcile our previous studies demonstrating increased transport affinity for amantadine in the presence of bicarbonate and decreased transport affinity in the presence of lactate.