Purpose. To demonstrate the in vivo transdermal delivery and establish the comparative pharmacokinetics of five β-blockers in hairless rat. Methods. Intravenous dosing was initially done via jugular cannula. For iontophoretic delivery, current (0.1 mA/cm2) was applied for 2 h through a drug reservoir patch containing the β-blocker (10 mg/ml). Blood samples were collected and analyzed by stereoselective HPLC assays. Any irritation resulting from patch application was quantified by a chromameter. Multilamellar liposomal formulation was prepared by the thin-film hydration method and converted to unilamellar liposomes by extrusion. Results. With transdermal iontophoresis, therapeutically relevant amounts of propranolol (83.78 ± 7.4 ng/ml) were delivered within an hour and lasted for up to 4 h. Cmax (185.1 ± 56.8 ng/ml) was reached at hour 3. A significantly higher amount (p < 0.05) of sotalol HCl was delivered compared to other β-blockers. There was no significant difference in the S/R ratio of AUC0-t for enantiomers after both intravenous and transdermal delivery. Skin irritation was significantly reduced (p < 0.05) when a liposomal formulation of the propranolol base was used rather than the base itself. Conclusions. The comparative pharmacokinetics of intravenous and transdermal iontophoretic delivery of five β-blockers in hairless rats was established. It was shown that there is no stereoselective permeation.