[3H]L-Adenosine, an enantiomer of the physiological D-adenosine, was shown previously to be taken up and released, at least in part, through nucleoside transporters in rat brain preparations. In the present study, we used clonal smooth muscle DDT1 MF-2 cells that contain almost exclusively equilibrative inhibitor-sensitive (es) nucleoside transporters to test the hypothesis that L-adenosine is a permeant for these bidirectional nucleoside transporters. DDT1 MF-2 cells accumulated approximately 3 times more [3H]D- than [3H]L-adenosine; 10 microM nitrobenzylthioinosine significantly (P < 0.01) inhibited the accumulation of [3H]D-adenosine by 86% and of [3H]L-adenosine by 63%. The IC50 values for the nitrobenzylthioinosine-sensitive portions of [3H]L- and [3H]D-adenosine accumulation were 1.6 and 2.0 nM, respectively. [3H]D-Adenosine accumulation was significantly (P < 0.05) inhibited by up to 72% with L-adenosine and [3H]L-adenosine accumulation was significantly (P < 0.01) inhibited by up to 52% with D-adenosine. Release of accumulated [3H]L-adenosine was temperature- and time-dependent, and was significantly (P < 0.05) reduced by 47% with dipyridamole, 39% with dilazep, and 45% with nitrobenzylthioinosine; the apparent IC50 value for nitrobenzylthioinosine was < 1 nM. These data indicate that a significant proportion of [3H]L-adenosine uptake and release in DDT1 MF-2 cells is mediated by es nucleoside transporters.