Background The cyanoacrylate follicular biopsy is an established method for the examination of the horny layer and quantitative assessment of microcomedones. We have optimized the method by separating follicular casts mechanically from the cyanoacrylate strips.Objectives To use this method to analyse topical therapy-induced changes of the lipid composition in the sebaceous follicular infundibulum.Methods Both the follicular casts and the residual skin surface strip, the last representing a mixture of stratum corneum and surface lipids, were extracted twice with n-hexane–ethanol under ultrasonication, evaporated, redissolved in chloroform–methanol and separated by high-performance thin layer chromatography, using cholesterol sulphate, cerebroside, ceramide types 3 and 4, cholesterol, oleic acid, triolein, cholesterol oleate and squalene as standards. Identification was performed by computer-assisted densitometric analysis. Six patient groups receiving adapalene 0·1%, tretinoin 0·025%, clindamycin 1%, clindamycin 1% + tretinoin 0·025%, benzoyl peroxide 5% or benzoyl peroxide 5% + erythromycin 2% were investigated before and 12 weeks after application.Results A significant decrease in free fatty acid proportions combined with an increase in triglycerides was observed in the groups receiving antimicrobial therapy, supporting the hypothesis of lipolysis due to microbial colonization. The groups treated with topical retinoids showed an additional significant increase in ceramide subfractions, most probably reflecting their influence on epidermal keratinization.Conclusions Our method proved suitable for the detection of quantitative and qualitative changes in lipid profiles of both infundibulum cast content and surface lipids. It enabled simple, non-invasive and objective assessment of the most relevant lipid classes in the sebaceous infundibulum, and efficient monitoring of drug effects on the follicular infundibulum.