The stratum corneum of the skin of patients with atopic dermatitis is highly susceptible to colonization by various bacteria, including Staphylococcus aureus. The defense system of the skin against bacterial invasion appears to be significantly disrupted in atopic dermatitis skin, but little is known about the defense mechanism(s) involved. As one sphingolipid metabolite, sphingosine is known to exert a potent antimicrobial effect on S. aureus at physiologic levels, and it may play a significant role in bacterial defense mechanisms of healthy normal skin. Because of the altered ceramide metabolism in atopic dermatitis, the possible alteration of sphingosine metabolism might be associated with the acquired vulnerability to colonization by S. aureus in patients with atopic dermatitis. In this study, we measured the levels of sphingosine in the upper stratum corneum from patients with atopic dermatitis, and then compared that with the colonization levels of bacteria in the same subjects. Levels of sphingosine were significantly downregulated in uninvolved and in involved stratum corneum of patients with atopic dermatitis compared with healthy controls. This decreased level of sphingosine was relevant to the increased numbers of bacteria including S. aureus present in the upper stratum corneum from the same subjects. This suggests the possibility that the increased colonization of bacteria found in patients with atopic dermatitis may result from a deficiency of sphingosine as a natural antimicrobial agent. As for the mechanism involved in the decreased production of sphingosine in atopic dermatitis, analysis of the activities of ceramidases, major sphingosine-producing enzymes, revealed that, whereas the activity of alkaline ceramidase did not differ between patients with atopic dermatitis and healthy controls, the activity of acid ceramidase was significantly reduced in patients with atopic dermatitis and this had obvious relevance to the increased colonization of bacteria in those subjects. Further, there was a close correlation between the level of sphingosines and acid ceramidase (r = 0.65, p <0.01) or ceramides (r = 0.70, p <0.01) in the upper stratum corneum from the same patients with atopic dermatitis. Collectively, our results suggest the possibility that vulnerability to bacterial colonization in the skin of patients with atopic dermatitis is associated with reduced levels of a natural antimicrobial agent, sphingosine, which results from decreased levels of ceramides as a substrate and from diminished activities of its metabolic enzyme, acid ceramidase.