Uridine transport was investigated in cultured chromaffin cells and plasma membrane vesicles from chromaffin tissue. In intact cells, the kinetic parameters for uridine uptake were K, 150 2 45 pM, and V,,, 414 2 17 pmol . lo6 cells-' . min-I. This low affinity for uridine and its inhibition by low concentrations of nitrobenzylthioinosine (K, 3 nM) and dipyridamole (K, 54 nM) are consistent with a facilitated diffusion nucleoside transport system. The IC,* value for the adenosine transport inhibition by uridine was very high (240 pM), agreeing with the relative affinities of these nucleosides in the chromaffin cell nucleoside transport system, which was 150-fold higher for adenosine than for uridine. Uridine was significantly metabolized in chromaffin cells but not in plasma membrane vesicles. The affinity of uridine transport measured in these membrane vesicles was reproducible and similar to the affinity found for intact cells with a K,,, value of 185 % 11 pM and a V,, value of 4.2420.10 pmol . mg protein-' . s-I. These membrane preparations were employed to investigate the regulatory action of ATP and other nucleotide analogues on nucleoside transport. ATP increased the V,,, value but the K, value was not significantly modified. Adenosine 5'-[p,y-imino]triphosphate, 1 ,W-ethenoadenosine 5'-triphosphate, and adenosine(5')-tetraphospho(5')adenosine (Ap,A) at 100 pM were able to mimic the ATP effect. These results agree with a regulatory role of ATP, and the uridine transport on chromaffin plasma membrane vesicles is a good model for analyzing the nucleoside-transporter function and regulation.A wide variety of mammalian cells have been reported to possess nucleoside transport systems [l]. The presence of high-affinity nucleoside transport mediated mainly by a facilitated-diffusion mechanism has been reported in neural cells and synaptosomal preparations. This system recognizes adenosine as the preferred endogenous substrate with K , values in the range 1-20 pM for this nucleoside [2-51. In neurochromaffin cells, which are a homogeneous neural cell population, the nucleoside transport mechanism has a high affinity for adenosine (K, 1 pM) [6]. This transport system was not dependent on a Na' gradient and was completely inhibited by low concentrations of nitrobenzylthioinosine (NPhCH,SIno; K, 0.01 nM), a specific inhibitor for the facilitated-diffusion systems [7, 81. The chromaffin cell model has proved to be very useful in studies of adenosine transport regulation. In recent studies, it was reported that activation of protein kinase A and protein Correspondence to M. Teresa Miras-Portugal,