The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [3H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with Km = 370 microM and Vmax = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na+ from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells (Km = 12 microM; Vmax = 0.34 nmol/min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N6-benzyladenosine. In decreasing order of potency, N6-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin.