The present study investigated the role of adenosine in the stimulatory action of paeoniflorin on in vitro glucose transport. Paeoniflorin increased the uptake of a radiolabeled, non-metabolizable glucose derivative into isolated white adipocytes of Wistar rat in a concentration-dependent manner and this action was abolished by the antagonist, 8-cyclopentyltheophylline, at concentrations sufficient to block the adenosine A 1 receptor. However, paeoniflorin failed to displace the binding of [3H]-8-cyclopentyl-1,3-dipropylxanthine in the isolated cerebrocortex of Wistar rat. Direct activation of the adenosine A 1 receptor does not seem to be responsible for the action of paeoniflorin. The stimulatory effect of paeoniflorin on radioactive glucose uptake was abolished in isolated rat white adipocytes pre-incubated with the adenosine deaminase at concentrations sufficient to metabolize endogenous adenosine. Mediation of endogenous adenosine in the action of paeoniflorin was further supported by the assay of adenosine released into the medium from rat white adipocytes incubated with paeoniflorin. These findings suggest that paeoniflorin could induce the release of adenosine from isolated rat white adipocytes and the released adenosine may activate the adenosine A 1 receptor to enhance glucose uptake.