Pyridostigmine bromide (PB) delivered transdermally may be a useful pretreatment for organophosphate poisoning. PB transdermal formulations were developed since this route has the potential to provide a more constant, prolonged and therapeutically-effective drug level in the body. Guinea pig skin sensitization studies, using a variation of the split adjuvant technique, were conducted with various PB transdermal formulations as part of a safety evaluation profile. 3 gel matrix formulations were tested. The 3 formulations contained 50% PB, 30% PB with 0.198% sodium lauryl sulfate (SLS) and 30% PB with 0.21% of a proprietary surfactant (PS), respectively. SLS and the proprietary surfactant were added to the formulations as dermal penetration enhancers. 9 groups of 10 animals were induced and challenged with 1 of the 3 PB or PB/surfactant formulations (3 groups per formulation). In addition, 2 groups of 10 animals were included in the study as positive controls that were induced and challenged with 2,4-dinitrochlorobenzene (DNCB). 44% of the animals responded positively at challenge to 50% PB. 80% of the animals responded positively at challenge to 30% PB/0.198% SLS and 82% of the animals had positive responses at challenge to 30% PB/0.21% PS. This study demonstrates that PB is a potential contact sensitizer that shows a potentiated response in the presence of surfactants.