Development of in vitro models to identify sensitizing chemicals receives public interest since animal testing should be avoided whenever possible. In this article we analyze two essential properties of sensitizing chemicals: skin penetration and dendritic cell (DC) activation. Activation of immature DC derived from peripheral blood monocytes was evaluated by flow cytometric analysis of CD86 positive cells and quantitative measurement of interleukin-1β and aquaporin P3 gene expression. The sensitizer 2,4,6-trinitrobenzenesulfonic acid induced a concentration-dependent response for all parameters, whereas the irritant sodium lauryl sulfate did not. When two related aromatic amines, p-toluylenediamine (PTD) and hydroxyethyl-p-phenylenediamine (HE-PPD) were tested, both induced substantial DC activation indicating their potential sensitizing properties. These findings contrasted with in vivo results: in murine local lymph node assays (LLNA) PTD, but not HE-PPD, was sensitizing using acetone/aqua/olive oil as vehicle. Skin penetration measurement revealed that this was due to bioavailability differences. On retesting HE-PPD in the LLNA using the penetration enhancer dimethylsulfoxide as vehicle, it induced a specific response. We conclude that in vitro analysis of DC activation capability of the two selected chemicals demonstrates that prediction of skin sensitization potential is possible provided that skin penetration data indicate sufficient bioavailability of the test compound.