Sphingosine is known to have potent biological activity, including pronounced anti-microbial action in vitro against Candida albicans and some bacteria. Several sphingosine bases are present in stratum corneum at concentrations several orders of magnitude above those in other tissues. Sphingosine forms an undissociated salt with organic sulfates, however, so that the free sphingosine in the epidermis may be inactivated by the cholesterol sulfate known to be present. To investigate this hypothesis, C. albicans was grown in cultures with graded concentrations of sphingosine added in ethanol. In 1% ethanol, 0.1-100 microgram/ml sphingosine completely prevented growth of the organism for 12 h. All cultures eventually entered log-phase growth and reached limiting density at a rate inversely proportional to sphingosine concentration. When sphingosine was added, together with an equimolar amount of cholesterol sulfate, there was no delay in the onset of growth of the yeast and the rate of growth and final density were similar to control cultures. These results demonstrate that natural ratios of cholesterol sulfate neutralize the anti-microbial activity of sphingosine in vitro. In the epidermis, endogenous cholesterol sulfate is hydrolyzed by sterol sulfatase at the skin surface, where the released sphingosine may resist microbial colonization of the stratum corneum. This mechanism for liberating anti-microbial sphingosine base only at the skin surface may protect the viable epidermis against known cytotoxic effects of free sphingosine.