Theophylline, caffeine, isobutylmethylxanthine, papaverine, N6-cyclohexyladenosine, N6-allyl-N6-cyclohexyladenosine ( ACHA ) and N6-L-phenylisopropyladenosine (L-PIA) inhibited the transport of adenosine, uridine and hypoxanthine in Novikoff rat hepatoma cells. The IC50 values for the inhibition of uridine transport ranged from 5 microM for ACHA to 3200 microM for caffeine and were inversely proportional to the lipid solubility of the inhibitors. L-PIA and papaverine inhibited uridine influx in a non-competitive manner, having a greater influence on the Michaelis-Menten constant than on maximum velocity, just as observed previously for the inhibition of nucleoside transport by dipyridamole and hypoxanthine. [3H]L-PIA rapidly accumulated in Novikoff cells at 25 degrees to about five times higher levels than present extracellularly. The initial rates of L-PIA uptake were directly proportional to its extracellular concentration between 0.01 and 240 microM and not affected by structurally related analogs, methylxanthines, papaverine, dipyridamole, or 2 mM uridine, but were dependent on temperature. We conclude that L-PIA inhibits nucleoside transport in these cells without being significantly transported by the carrier, that it equilibrates rapidly across the plasma membrane without carrier mediation consistent with its lipophilicity, and that it accumulates concentratively in cells due to partitioning into membrane lipids and binding to intracellular components.