Confocal laser scanning microscopy (CLSM) is a well-used microscopic tool that provides valuable morphological and functional information within cells and tissues. The application of CLSM to skin and the topical penetration of nanoparticles (NP) will be addressed. First, we describe the advantages of confocal microscopy compared to other techniques and its use relative to skin research. Second, we discuss the ability of CLSM to detect single NP. Regarding their interaction with skin, the appropriate method to retain nanoparticle localization in the tissue with minimal fixation is critically important. Also, the interaction of several different types of NP (quantum dots, fullerene and dendrimers) and their interaction with skin detected by CLSM under various conditions (flexed, tape stripped and abraded skin) is reviewed. Finally, human epidermal keratinocytes and dendritic cells that serve as appropriate in vitro models for skin cell interactions and cellular uptake of NP are also discussed. In conclusion, the unique functions of CLSM such as the ability to detect fluorescence, optical sectioning, three dimensional remodeling, as well as its use in the reflection mode in tandem with other methods, provides great promise with broad applications regarding the interactions of nanomaterials with skin. (C) The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.