The aim of this study was to investigate the intranasal absorption of R-(+)-WIN 55,212-2 mesylate in vivo and in vitro. Permeation experiments of R-(+)-WIN 55,212-2 formulations with 2% dimethyl-β-cyclodextrin (DMβCD), 2% trimethyl-β-cyclodextrin (TMβCD) or 2% randomly methylated-β-cyclodextrin (RAMβCD) in 1:1 propylene glycol/saline and 1.5% propylene glycol + 3% Tween 80 in saline were conducted using EpiAirway™ tissue and an anesthetized rat nasal absorption model, respectively. Samples were analysed by liquid chromatography-mass spectrometry. Mucosal tolerance was screened using paracellular marker permeation and tissue viability as indices. Nasal absorption of WIN 55,212-2 was rapid, with a tmax (time of peak concentration) of 0.17 to 0.35 h in vivo. Relative to 1.5% propylene glycol + 3% Tween 80 (control), 1:1 propylene glycol/saline, RAMβCD, DMβCD and TMβCD resulted in 24-, 20-, 17- and 10-fold WIN 55,212-2 permeation increases in vitro, respectively. The in vivo absolute bioavailabilities were also increased with 1:1 propylene glycol/saline, RAMβCD, DMβCD and TMβCD compared to 1.5% propylene glycol + 3% Tween 80 (0.15 vs. 0.66-0.77). The viability of the EpiAirway™ tissues was significantly reduced by DMβCD and TMβCD formulations. This study showed that WIN 55,212-2 mesylate can be delivered via the nasal route. Absorption of R-(+)-WIN 55,212-2 was rapid and bioavailability was significantly improved using methylated cyclodextrins and propylene glycol-based cosolvent.