Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi – India The present study was undertaken to assess passive and iontophoretic transport of Timolol maleate (TM) across rat abdominal skin, pretreated with different permeation enhancers, alone and in combination. Freshly excised full thickness abdominal skin of rat (rat skin) was pretreated with permeation enhancer(s) for 6 hours. Sodium lauryl sulphate (SLS), dimethyl sulfoxide (DMSO), and their different combinations were used as permeation enhancers. Anodal iontophoresis (anode in donor solution) was performed using current density of 0.5 mA/cm2. Iontophoretic transport through untreated full thickness rat abdominal skin was greater than passive diffusion (Enhancement factor = 17.09). The pretreatment of rat skin with permeation enhancers resulted in enhanced passive and iontophoretic steady state flux. Rat skin pretreated with combination of 30 % w/v DMSO and different concentration of SLS significantly (P<0.05) enhanced the passive and iontophoretic flux of TM in comparison to when these enhancers were used alone. The skin pretreated with a combination of 30 % w/v DMSO and 0.3 % w/v SLS showed highest flux of TM during iontophoresis (Enhancement factor = 93.47). Microphotographs of rat skin pretreated with combination of 30 % w/v DMSO and 0.3 % w/v SLS during iontophoresis reveals that enhancement in flux may be due to swelling and disruption of epidermal layer. Keywords : TRANSDERMAL, IONTOPHORESIS, TIMOLOL MALEATE, PERMEATION ENHANCERS