Penetration enhancers are often used as additives in pharmaceutical and dermatological preparations. It should be expected that in many cases penetration enhancers not only enter the stratum corneum but also reach the viable cells of the epidermis and exert a toxic effect. This study focused on a series of well-known compounds that are often used as skin penetration enhancers; namely, ethanol, propylene glycol, dimethylsulfoxide, dimethylformamide, and Brij 96. In order to obtain more insight in the potential skin toxicity of these agents, they were administrated to cultured human keratinocytes and fibroblasts and the following cytotoxicity assays were performed: inhibition of the proliferation of fibroblasts and keratinocytes; inhibition of collagen contraction by fibroblasts; and cell morphology changes in confluent cultures of fibroblasts and keratinocytes. In all assays performed, the same trend was observed: ethanol was the least toxic, propylene glycol, dimethylsulfoxide, and dimethylformamide were moderately potent, and Brij 96 was the most toxic agent. An obvious advantage of the in vitro model presented here is its immediate availability and reproducibility, which allows for the comparison of a large series of topical agents (e.g., penetration enhancers) with respect to their cell toxicity under standardized conditions. However, this single-cell model lacks some of the properties found in intact skin, such as the stratum corneum barrier, and interactions between keratinocytes and other cells, such as Langerhans cells. Hence, extrapolation of these data to in vivo should be done with caution