This study evaluated a model of skin permeation to determine the depth of delivery of chlorhexidine into full-thickness excised human skin following topical application of 2% (wt/vol) aqueous chlorhexidine digluconate. Skin permeation studies were performed on full-thickness human skin using Franz diffusion cells with exposure to chlorhexidine for 2 min, 30 min, and 24 h. The concentration of chlorhexidine extracted from skin sections was determined to a depth of 1,500 m following serial sectioning of the skin using a microtome and analysis by high-performance liquid chromatography. Poor penetration of chlorhexidine into skin following 2-min and 30-min exposures to chlorhexidine was observed (0.157 ؎ 0.047 and 0.077 ؎ 0.015 g/mg tissue within the top 100 m), and levels of chlorhexidine were minimal at deeper skin depths (less than 0.002 g/mg tissue below 300 m). After 24 h of exposure, there was more chlorhexidine within the upper 100-m sections (7.88 ؎ 1.37 g/mg tissue); however, the levels remained low (less than 1 g/mg tissue) at depths below 300 m. There was no detectable penetration through the full-thickness skin. The model presented in this study can be used to assess the permeation of antiseptic agents through various layers of skin in vitro. Aqueous chlorhexidine demonstrated poor permeation into the deeper layers of the skin, which may restrict the efficacy of skin antisepsis with this agent. This study lays the foundation for further research in adopting alternative strategies for enhanced skin antisepsis in clinical practice.Effective skin antisepsis is essential in preventing infections associated with invasive procedures, such as intravascular catheter insertion or surgery. A range of skin antiseptic agents are available in the clinical setting, such as povidone-iodine and chlorhexidine compounds at various concentrations with alcoholic or aqueous solutions. However, a 2% (wt/vol) chlorhexidine solution is the recommended agent to be used prior to invasive procedures according to the EPIC (evidence-based practice in infection control) and CDC guidelines (18,19). Two percent chlorhexidine digluconate (CHG) has been shown to significantly reduce intravascular catheter-related infections (14), yet 2% (wt/vol) CHG in 70% (vol/vol) isopropyl alcohol demonstrates activity superior to that of aqueous CHG solution in a preoperative skin preparation (9) and in vitro carrier tests (1). However, little is known about the kinetics of chlorhexidine skin permeation from either of these solutions (11,25). Microorganisms colonizing the skin not only reside on the skin surface but are also found to inhabit hair follicles and lower skin depths (8). Many antimicrobial agents exhibit restricted permeation of the skin (8) and fail to reach the deeper layers, including the hair follicles, which harbor coagulasenegative staphylococci (2,7,8,13,15) and propionibacteria (13). Commensal microorganisms may therefore persist at the site of incision following skin antisepsis (4, 22), and such resident organisms may cause infec...