Rabbit central nervous system (CNS) preparations have been used to study the central effects of adenosine, but little is known about the specific uptake mechanisms in rabbit brain involved in the regulation of extracellular adenosine concentrations. The present study assessed the kinetic and pharmacological characteristics of the uptake of [3H]uridine (a poorly metabolized substrate for adenosine transporters) by rabbit cortical synaptosomes, to define the transporter subtypes involved and to evaluate species variability in transporter characteristics. [3H]Uridine transport into rabbit cortical synaptosomes was mediated by two saturable, facilitated diffusion systems with characteristics compatible with the es and ei transporter subtypes identified in other mammalian species. About 65% of the total transport was mediated by the es system, and Km estimates of 320 and 94 microM were determined for [3H]uridine uptake by the es and ei transporter, respectively. These results differ significantly from the subtype ratio and kinetic characteristics reported for rat and guinea pig cortical synaptosomes, where most of the transport was mediated by an ei subtype. Dipyridamole, dilazep, nitrobenzylthioinosine, R75231, soluflazine, and mioflazine were relatively more effective as inhibitors of es-mediated uptake (compared with ei), while the substrates adenosine, cytidine, and guanosine did not distinguish between the es and ei transporters in rabbit cortical synaptosomes. These results highlight the significant species-tissue variability in nucleoside transporter characteristics and subtype expression, and emphasize the need to characterize the transporters in human CNS tissue to allow the rational development of CNS-active therapeutics based on inhibition of nucleoside transport.