The aim of the present investigation was to evaluate the percutaneous absorption of tolterodine (TOL) using O-acylmenthol derivatives as enhancers as well as to correlate their enhancing activity under in vitro and in vivo conditions. The in vitro permeation studies of TOL were conducted in isopropyl myristate (IPM) solution or from patches in side-by-side diffusion cells. TOL pharmacokinetic parameters were determined after intravenous administration and topical application of patches without enhancer or with l-menthol and (E)-2-isopropyl-5-methylcyclohexyl octadec-9-enoate (M-OA) as enhancers in rats. The in vitro permeation studies indicated that M-OA was the most promising enhancer for transdermal delivery, as 2-isopropyl-5-methylcyclohexyl 2-hydroxypanoate (M-LA) was merely effective in IPM solution. There was no significant difference between the control and l-menthol group in terms of the flux before patches were removed, while the skin reservoir effects of the enhancer-containing groups were significantly greater than that of the control group. The mean steady-state plasma concentrations after applying patches without enhancer or with l-menthol and M-OA as enhancers were 0.89, 0.84 and 1.47 microg/mL, respectively. The in vivo results observed from the three types of patches in rats were in good agreement with the plasma concentrations predicted from the in vitro data.