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Summary Background  Acne vulgaris is a disorder of the sebaceous follicles. Propionibacterium acnes can be involved in inflammatory acne. Objectives  This case-control study aimed at investigating the occurrence and localization of P. acnes in facial biopsies in acne and to characterize the P. acnes phylotype in skin compartments. Methods  Specific monoclonal and polyclonal antibodies were applied to skin biopsies of 38 patients with acne and matching controls to localize and characterize P. acnes and to determine expression of co-haemolysin CAMP factor, a putative virulence determinant. Results  Follicular P. acnes was demonstrated in 18 (47%) samples from patients with acne and eight (21%) control samples [odds ratio (OR) 3·37, 95% confidence interval (CI) 1·23-9·23; P = 0·017]. In 14 (37%) samples from patients with acne, P. acnes was visualized in large macrocolonies/biofilms in sebaceous follicles compared with only five (13%) control samples (OR 3·85, 95% CI 1·22-12·14; P = 0·021). Macrocolonies/biofilms consisting of mixed P. acnes phylotypes expressing CAMP1 were detected in both case and control samples. Only four samples tested positive for the presence of Staphylococcus spp. and fungi were not observed. Conclusions  We have for the first time visualized different P. acnes phylotypes in macrocolonies/biofilms in sebaceous follicles of skin biopsies. Our results support the hypothesis that P. acnes can play a role in the pathogenesis of acne as acne samples showed a higher prevalence of follicular P. acnes colonization, both in terms of follicles containing P. acnes and the greater numbers of bacteria in macrocolonies/biofilms than in control samples.