Pimecrolimus and tacrolimus are calcineurin inhibitors used for the topical treatment of atopic dermatitis. Although structurally similar, they display specific differences including higher lipophilicity and lower skin permeation of pimecrolimus. The aim of the present study was to understand the reason for the differences in skin permeation; in addition, plasma protein binding of the two drugs was analyzed side by side as a basis for comparison of systemic exposure to free drug. Permeation of pimecrolimus and tacrolimus through a silicon membrane was found to be similar; therefore, we assumed that differences in skin permeation could be caused by differences in affinity to skin components. To test this hypothesis, we investigated binding of pimecrolimus and tacrolimus to a preparation of soluble human skin proteins. One binding protein of approximately 15 kDa, probably corresponding to macrophilin12, displayed a similar binding capacity for pimecrolimus and tacrolimus. However, less specific, nonsaturating binding to other proteins was approximately 3-fold higher for pimecrolimus. Because of the high local drug concentration after topical administration, the unspecific, high-capacity binding is probably dominating the permeation through skin. In plasma both drugs bound predominantly to lipoproteins, which may affect disposition differently from albumin binding. The unbound fraction of pimecrolimus in human plasma was approximately 9-fold lower compared with that of tacrolimus (0.4 +/- 0.1 versus 3.7 +/- 0.8%). In conclusion, these results provide an explanation for the observed lower systemic exposure to pimecrolimus than to tacrolimus after topical application and suggest that differences in systemic exposure to free drug might be even more pronounced.